ArticlePDF AvailableLiterature Review

Genetics and Biochemistry of Phenol Degradation by Pseudomonas sp. CF600

January 1995
Biodegradation 5(3-4):219-36

January 1995
5(3-4):219-36

DOI:10.1007/BF00696461

Source
PubMed

Authors:

Victoria Shingler

Umeå University

Pseudomonas sp. strain CF600 is an efficient degrader of phenol and methylsubstituted phenols. These compounds are degraded by the set of enzymes encoded by the plasmid located dmpoperon. The sequences of all the fifteen structural genes required to encode the nine enzymes of the catabolic pathway have been determined and the corresponding proteins have been purified. In this review the interplay between the genetic analysis and biochemical characterisation of the catabolic pathway is emphasised. The first step in the pathway, the conversion of phenol to catechol, is catalysed by a novel multicomponent phenol hydroxylase. Here we summarise similarities of this enzyme with other multicomponent oxygenases, particularly methane monooxygenase (EC 1.14.13.25). The other enzymes encoded by the operon are those of the well-known meta-cleavage pathway for catechol, and include the recently discovered meta-pathway enzyme aldehyde dehydrogenase (acylating) (EC 1.2.1.10). The known properties of these meta-pathway enzymes, and isofunctional enzymes from other aromatic degraders, are summarised. Analysis of the sequences of the pathway proteins, many of which are unique to the meta-pathway, suggests new approaches to the study of these generally little-characterised enzymes. Furthermore, biochemical studies of some of these enzymes suggest that physical associations between meta-pathway enzymes play an important role. In addition to the pathway enzymes, the specific regulator of phenol catabolism, DmpR, and its relationship to the XylR regulator of toluene and xylene catabolism is discussed.

. Summary of the genes and gene products of the dmp operon.

…

. Compilation of sequence homologies between the recta-cleavage pathway proteins of the dmp a-, xyl b-, tod c-and bph a-systems.

…

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Biodegradation

5: 219-, 1994. 219

(g) 1994

Kluwer Academic Publishers. Printed in the Netherlands.

Genetics and biochemistry of phenol degradation by

Pseudomonas

sp. CF600

Justin Powlowski 1 & Victoria Shingler 2

1 Department of Chemistry and Biochemistry, Concordia University, 1455 de Maisonneuve Boulevard West,

Montreal, Quebec, Canada H3G 1MS; 2 Department of Cell and Molecular Biology, Umed University, S-901 87

Umed, Sweden

Received 20 September 1993; accepted 31 January t994

Key words:

Phenols, oxygenases,

meta-cleavage

pathway, dehydrogenases,

Pseudomonas

Abstract

Pseudomonas

sp. strain CF600 is an efficient degrader of phenol and methylsubstituted phenols. These compounds

are degraded by the set of enzymes encoded by the ptasmid located

dmpoperon.

The sequences of all the fifteen

structural genes required to encode the nine enzymes of the catabolic pathway have been determined and the

corresponding proteins have been purified. In this review the interplay between the genetic analysis and biochemical

characterisation of the catabolic pathway is emphasised. The first step in the pathway, the conversion of phenol

to catechol, is catalysed by a novel multicomponent phenol hydroxylase. Here we summarise similarities of

this enzyme with other nmlticomponent oxygenases, particularly methane monooxygenase (EC 1.14.13.25). The

other enzymes encoded by the operon are those of the well-known

meta-cleavage

pathway for catechol,

and

include the recently discovered

meta-pathway

enzyme aldehyde dehydrogenase (acylating) (EC t.2.1.I0). The

known properties of these

meta-pathway

enzymes, and isofunctional enzymes from other aromatic degraders, are

summarised. Analysis of the sequences of the pathway proteins, many of which are unique to the

meta-pathway,

suggests new approaches to the study of these generally little-characterised enzymes. Furthermore, biochemical

studies of some of these enzymes suggest that physical associations between

recta-pathway

enzymes play an

important role. In addition to the pathway enzymes, the specific regulator of phenol catabolism, DmpR, and its

relationship to the XylR regulator of toluene and xylene catabolism is discussed.

Introduction

Utilisation of phenol as a source of microbial nourish-

ment has perhaps been overshadowed by awareness of

its generally unhealthy effects on humans. Although

the use of phenol as an antiseptic for treating wounds

and wound dressings was of unquestionable value in

helping to establish the importance of sterile tech-

niques in medicine, and probably helped save the lives

of many patients, the toxicity of the compound was

evident from, amongst other things, its contemporane-

ous use as a popular orally-administered suicidal agent

(Lister 1867; McGrew 1985). While the use of phenol

as an antiseptic dwindled by the turn of the century,

its application as a general disinfectant continued, as

did development of new uses for phenol and its deriva-

tives. Today approximately 1700 million kilograms of

phenol are synthesised annually in the United States

alone (Anonymous 1993), and countless quantities of

phenol and its derivatives are produced as industrial

by-products.

Considering the large scale on which this highly

toxic compound and related chemicals are produced,

its degradability is of obvious interest. Persistence of

phenol itself in the environment is not a particular prob-

lem, with both physico-chemical and biological agents

contributing to its degradation (Howard et al. 1991).

However, as with any metabolic process, biodegrad-

ability can be affected by many variables, including,

for example, the presence of alternative carbon sources

or substituents on the phenol ring. While such limita-

tions may be overcome by genetic manipulation of

the catabolic pathway involved, dissimilatory mecha-

nisms and their regulation must first be characterized

220

at the molecular level in individual microbial species.

Since phenol is a relatively simple, unadorned, aromat-

ic compound for which much information on biodegra-

dation is already available, phenol-degrading bacteria

provide good model systems for these kinds of stud-

ies. Microbial phenol degradation is unlikely to be a

recently-acquired trait. While the use of phenol on a

large scale by humans is relatively recent, microbes

have long been exposed to biologically generated phe-

nols and methyl-substituted phenols produced, for

example, by enterobacteria from tyrosine (Kumagai

et al. 1970; Spoelstra 1977) and by the break-down

of plant materials. The isolation of phenol-degrading

bacteria was achieved as early as 1932 (Happold &

Key 1932), and numerous others have been unearthed

since then. While many of the most-studied phenol

degraders are pseudomonads, other bacterial genera,

such as Bacillus, as well as yeast, are also represent-

ed (Gurujeyalakshmi & Oriel 1989; Neujahr & Gaal

1973).

Work during the nineteen sixties and seventies with

phenol-degrading Pseudomona putida U was respon-

sible for much of the progress in understanding the

biochemistry of the meta-cleavage pathway (reviewed

in Bayly & Barbour 1984). In this pathway, ring cleav-

age adjacent to two aromatic hydroxyls (Fig. 1) is

the distinguishing feature, with the subsequent series

of reactions dictated by the nature of the ring-fission

product. This contrasts with the ortho-cleavage path-

way, used by some phenol degraders, in which cate-

chol (1,2-dihydroxybenzene) produced from phenol is

cleaved between the catechol hydroxyls, with an entire-

ly different set of enzymes required for metabolism

of the distinctive ring-cleavage product (reviewed by

Ornston & Yeh 1982). One consequence of the enzy-

mologies of the two pathways is that while the ortho-

cleavage pathways of pseudomonads cannot tolerate

methyl-substituents, all three mono-methyl substitut-

ed phenols can be degraded via the meta-cleavage

pathway. Ortho-cleavage degraders can, however, cir-

cumvent this problem by first oxidizing the methyl

group to a carboxyl, and then using a separate set of

enzymes for the resulting hydroxybenzoic acid (see

Dagley 1986).

The main emphasis in this review is work done

on the dmp system which encodes the phenol degra-

dation pathway of Pseudomonas sp. strain CF600.

In addition to phenol, this organism metabolizes all

monomethylphenols, as well as 3,4-dimethylphenol,

via the dmp-encoded hydroxylation and meta-cleavage

pathway enzymes. This review highlights the interplay

between the analysis of the genes of this and other

pathways, and the biochemical studies of the enzymes

involved.

Genetic location and organisation of the dmp

system

Like many other pathways for the catabolism of aro-

matic compounds, the phenol/dimethylphenol (dmp)

pathway of Pseudomonas sp. strain CF600 is plasmid

encoded. The plasmid, designated pVI150, belongs to

the P-2 incompatibility group (Shingler et al. t989).

Like two other IncP-2 catabolic plasmids, OCT and

CAM (Fennewald et al. 1978; Rheinwald et al. t973),

pVI150 is very large, and exceeds 200 kb in size. Due to

the large size of pVI 150 and attendant difficulties in its

purification, initial isolation of the pathway-encoding

DNA necessitated utilization of methods more often

associated with isolation of chromosomally-encoded

genes (Shingler et al. 1989; Bartilson et al. 1990).

The results of genetic dissection of the 15 kb DNA

region encoding the dmp system, and the locations

of the sixteen genes involved, are summarised in Fig.

1. This figure also illustrates the biochemical route,

via hydroxylation and a meta-cleavage pathway, for

the catabolism of the pathway substrates. The fifteen

genes encoding the enzymes of the pathway (Table

1) are clustered in the dmp operon. Our attention ini-

tially focused on the DNA encoding the first enzyme

of the pathway, phenol hydroxylase, that catalyses

the conversion of phenol to catechol. DNA encod-

ing this enzyme activity was distinguished from that

encoding the remaining pathway enzymes by express-

ing different parts of the dmp operon in a strain of

Pseudomonas that possesses an ortho-cleavage path-

way capable of further catabolism of catechol, but

not methylcatechols. Subsequent testing of the growth

range of the resulting strains functionally defined the

DNA tbr the phenol hydroxylase (growth on phenol

only) and the entire pathway (growth on phenol and

methylphenols).

A combination of nucleotide sequencing and

polypeptide analysis identified six genes, dmp-

KLMNOP, and their corresponding products, P0, P1,

P2, P3, 174 and P5, within the phenol hydroxylase-

coding region. Deletions within each gene in turn, and

expression of each individual open reading frame, cou-

pled with polypeptide analysis, confirmed the assign-

ment of the polypeptide products to individual genes

(Nordlund et al. 1990a). As discussed below, the find-

Bg P B Bg P P N P E N

I I. I I II. I

I. I.

i 2 ~ i ~ 6 ~ h 9 10 fl 12 is f,

| l I u • II • I I II • • • • I

R K L M N 0 P Q B C D E F (3 H

[ Phenol Hydroxylase

M~m -Pathway Enzymes

221

15 kb

dmp(K)LMNOP

dmp(Q)B dmpD dmpE dmpG drapF

NADH, H +. q ~H /CHO H I"120 ~H 2 C~OH HzO CH 3 COOH (~,-I 3 NAD +. CoASH Ve~I3

k >

.o o --x--"

,usa2 - ,L

0 ~ NADH, H 0 ~ SCoA

, ~ "OH HCOOH v

+ o

N~r~ n" ~ ampc

o ~ "COOH

~/cOOHcooH dmpI CO0~ooH

OH ~

~(0

Fig. 1.

Schematic representation of the phenol/dimethylphenol pathway encoded by a 15 kb region of the pVI150 catabolic plasmid of

Pseudomonas

sp. strain CF600. The arrows indicate the divergent transcription of the

dmpR

regulatory gene and the

dmpKLMNOPQBCDE-

FGHI-operon.

Enzyme functions are as shown in Table 1. Both the hydrolytic

(dmpD-encoded)

and the 4-oxalocrotonate

(DmpCIH-encoded)

branches of the

meta-cleavage

pathway are shown. Restriction enzyme recognition site are:

B, BamHI; Bg, BgllI; E, EcoRI; N, NotI; P,

PvulI.

Table 1.

Summary of the genes and gene products of the

dmp

operon.

Gene Amino acid Molecular mass (kDa) Product Function Reference

residues predicted/estimated

dmpK

92 10.6/12.5 P0

dmpL

331 38.2/34.0 P1

dmpM

90 10.5/10.0 P2

dmpN

517 60.5/58.0 P3

dmpO

119 13.2/13.0 P4

dmpP

353 38.5/39.0 P5

dmpQ

112 12.2/12.0 DmpQ

dmpB

307 35.2/32.0 C230

dmpC

486 51.7/50.0 HMSD

dmpD

283 31.0/30.0 HMSH

dmpE

261 27.9/28.0 OEH

dmpF

312 32.7/35.0 ADA

dmpG

345 37.5/39.0 HOA

dmpH

264 28.4/28.5 4OD

dmpl

63 7.1/6.7 401

Unknown

Phenol hydroxylase component

Ferredoxin-like protein

Catechol 2,3-dioxygenase, EC 1.13.11.2

2-Hydroxymueonic semialdehyde,

dehydrogenase, EC 1.2.1 .-

2-Hydroxymuconie semialdehyde hydrolase

2-Oxopent-4-dienoate hydratase, EC 4.2.1.80

Aldehyde dehydrogenase (acylating), EC 1.2.1.10

4-Hydroxy-2-oxovalerate aldolase, EC 4.1.3.-

4-Oxalocrotonate decarboxylase, EC 4.1.1 .-

4-Oxalocrotonate isomemse, EC 5.3.2.-

Nordlund et al. 1990

Nordlundet al. 1990

Nordlund et al. 1990

Nordlnnd et al. 1990

Shingler et al. 1992

Bartilson & Shingler 1989

Nordlund & Shingler 1990

Shingler et al. 1992

ing that the phenol hydroxylase encoded by pV1150 is

multicomponent was initially surprising and prompted

further studies of this unusual enzyme.

The remaining nine genes of the operon,

dmpQBCDEFGHI,

encode the

meta-cleavage

pathway

enzymes for conversion of catechol to pyruvate and

acetyl-CoA (Table 1, Fig. 2). Assignment of a func-

tion to each of these genes was achieved using a com-

bination of expression of open reading frames, both

alone and in combination, nucleotide sequencing, and

222

sion of growth substrates to catechols appear to be clus-

tered in a single operon together with the meta-pathway

genes. However, the bph and tod operons only code for

an abridged version of the meta-cleavage pathway and

do not include genes for the 4-oxalocrotonate branch

(see Table 2), which is not necessary for the metabo-

lites of these two pathways. Comparisons between all

of these pathways and the dmp-encoded meta-cleavage

pathway will be included. However, before discussion

of the meta-cleavage pathway, the dmp-encoded phe-

nol hydroxylase that feeds it will be considered.

Initial hydroxylation of phenol

Fig.2. Proteinsencodedbythedmpoperon.2to 5 ~gofprotein were

analysed on a gradient ( 10 to 20%) SDS polyacrylamide gel. Lane 1,

DmpK (P0); 2, DmpLNO (P1 -P3-P4); 3, DmpM (P2); 4, DmpP (P5);

5, DmpQ; 6, DmpB (C230); 7, DmpC (HMSD); 8, DmpD (HMSH);

9, DmpEH (OEH/4OD); 10, DmpFG (ADA/HOA); I 1, Dmpl (4OI):

Molecular mass standards are given in kDa on the right-hand side.

All polypeptides were purified from phenol-grown

Pseudomonas

sp. strain CF600 except DmpK (lane 1), DmpQ (lane5) and DmpC

(lane7), which were purified from separate E. coli strains express-

hag the corresponding genes. Enzyme abbreviations and estimated

molecular masses are given in Table 1.

biochemical techniques (Bartilson & Shingler 1989;

Nordlund & Shingler 1990; Shingler et al. 1992).

Since many pathways for the catabolism of aro-

matic compounds include meta-cleavage pathway

enzymes, the dmp genes and proteins have counterparts

in other systems. The most extensively studied of these

are those from pseudomonads harbouring the pWW0-

encoded toluene/xylene xyl pathway, the NAH7- and

pWW60-1-eneoded naphthalene/salicylate nal (sal)

pathways, and the phenol degradation pathway of P.

putida U. All these systems have both branches of

the meta-cleavage pathway as depicted in Fig. 1 (see

Assinder & Williams 1990; Williams & Sayer 1994 for

recent reviews). Using gene-specific probing, this has

also been shown to be true for the meta-pathway genes

of the Pseudomonas sp. strain IC biphenyl degradation

pathway (Carrington et al. 1994). Recently, nucleotide

sequences have been completed for two chromosoma-

lly encoded meta-cleavage pathways involved in the

catabolism of (chloro-)biphenyl and toluene respec-

tively: the Pseudomonas sp. strain KKS 102 bph system

(Kikuchi et al. I993), and the P. putida F1 rod systern

(Lau et al. 1993). In these two systems, like that of

Pseudomonas sp. strain CF600, the genes for conver-

Although some of the first well-studied bacterial

aromatic degraders included phenol-degrading pseu-

domonads that hydroxylate the ring as aprelude to ring-

cleavage, little biochemical information about phe-

nol hydroxylases from these bacteria was published.

Work that required meagurements of phenol hydrox-

ylase activity relied on oxygen-uptake experiments

using whole cells. In fact, the first reported purification

of a hydroxylase specific to a microbial phenol degra-

dation pathway was for phenol hydroxylase from the

yeast Trichosporon cutaneum (Neujahr & Gaal 1973).

This enzyme is a flavoprotein hydroxylase with prop-

erties similar to those of many bacterial flavoprotein

hydroxylases used for catabolism of such compounds

asp-hydroxybenzoate and melilotate (reviewed by Bal-

lou 1982) (Fig. 3).

All of these flavoprotein hydroxylases mono-

oxygenate the aromatic ring at a position ortho (or

in some cases para) to a pre-existing hydroxyl group.

The role of the flavin in these enzymes is the activa-

tion of molecular oxygen, which is normally unreactive

with organic compounds, Activation initially involves

reaction of 2-dectron reduced flavin with 02to form a

flavin 4a-hydroperoxide, which is an electrophilic, rel-

atively weak, oxygenating agent (Entsch et al. 1976).

Oxygenation by this enzyme-bound species is aided

by delocalization of electrons into the ring from the

hydroxyl group of the substrate. Conversion of pheno-

lies to dihydroxylated ring-fission substrates by pro-

teins containing flavin as the sole prosthetic group is

thus a well-documented occurrence in microbial aro-

matic metabolism.

This contrasts with oxygenases for compounds,

such as benzene, toluene, or phthalate, that are less

susceptible to oxygenation by virtue of the lack of an

electron-donating substituent on the ring. For these

compounds, a more potent enzyme-generated oxy-

223

Table 2.

Compilation of sequence homologies between the

recta-cleavage

pathway proteins of the

dmp a-, xyl b-, tod c- and

bph a-systems.

Ferredoxin-like proteins DmpQ

2,3-dioxygenases, EC 1. t3.11 .- DmpB

Dehydrogenases (HMSD), EC 12.I.- DmpC

Hydrolases (HMSH) DmpD

Hydratases (OEH), EC 4.2,I.80 DmpE

CoA-dependent dehydrogenases (ADA), EC 1,2.1.10 DmpF

Aldolases (HOA), EC 4.1.3.- DmpG

Decarboxylases (4OD), EC 4.1.1.- DmpH

Isomerases (4OI), EC 5.3.2.- DmpI

XylT (64) -

XylE (84) TodE (22) BphC (22)

XylG (84) -

XylF (75) TodF (64) BphD (32)

XylJ (89) TodG (41) BphE (43)

XylQ (90) TodI (75) BphG (78)

XytK (87) TodH (78) BphF (81)

XylI (89) -

XylH (78) -

TodE/BphC (50)

The percent identity of the deduced amino acid sequences when compared with those of the

drop

system are shown in

parenthesis.

a The phenol/dimethylphenol pVl 150-encoded pathway of

Pseudomonas

sp. CF600: references as in Table 1.

b The toluene/xylene pWW0-encoded pathway of

Pseudomonasputida

mt-2: Nakai et al. (1983), Harayama et al. (1991),

Harayama & Rekik (1993), Horn et al. (1991), Chen et al. (1992).

c The chromosomally-encoded toluene pathway of

Pseudomonasputida

F1: Zylstra & Gibson (1989), Menn et al. (1991),

Lau et al. (1993) and Lau (pers. comm.).

a The chromosomally-encoded (chloro-)biphenyl pathway of

Pseudomonas

sp. KKS102: Kimbara et al. (l 989), Kikuchi et

al. (1993).

Homologies not determined in the above references were analysed using the GCG Wisconsin sequence analysis software

package .2

MONOOXYGENASES DIOXYGENASES

NADPH 2

phenol

NADP,,.~ ~t, j/~k~l~

hydroxylase

(I. cutaneum)

t1~-11

NADH2

H methane

H~~H

H~O OH

NADH2~

NAP"

4mothoxy n to NADH2

p eno, NAo' C

hydroxylase

(Pseudomonas CF600)

phthalate

dioxygenase

benzoate

dioxygenase

toluene

(or biphenyl)

dioxygenase

naphthalene

dioxygenase

Fig. 3.

Composition of representative hydroxylating mono- and di-oxygenases involved in microbial metabolism. All are

Pseudomonas

enzymes except phenol hydroxylase (T.

cutaneum),

and methane monooxygenase

(M. capsulatus

(Bath),

M. trichosporium).

Subscripts denote

ferredoxin (Fd) or Reiske (R)- type 2Fe-2S eentres. Original references may be found in: Mason & Cammack (1992), Haryama et al. (1992),

and Neujahr & Gaal (1973).

224

genating agent is required. This is commonly thought

to be generated by complexation of 02 with iron in

some form at the active site of the enzyme (Fig. 3),

although in most cases shown here the nature of the

activated oxygen species is poorly defined. One or

more electron-transferring proteins are associated with

the oxygenating component of each enzyme in order to

shunt electrons from NAD(P)H for complete reduction

of 02 (Fig. 3). While these enzyme systems dioxy-

genate, some examples of multicomponent monooxy-

genases used for unactivated non-aromatic compounds

are also shown in Fig. 3.

In the light of the above, it was quite surprising to

find that Pseudomonas sp. strain CF600 elaborates a

multicomponent phenol hydroxylase that has little in

common with single-component flavoprotein hydrox-

ylases. As will be discussed below, while flavopro-

tein phenol hydroxylases are now known to be used by

some other phenol-degrading species of Pseudomonas,

the multicomponent variety is not restricted to Pseu-

domonas sp. strain CF600.

Phenol hydroxylase: A new aromatic oxygenase

As described earlier, the dmpKLMNOP gene products,

P0 to P5, were all found to be essential for growth of the

catechol-degrading test strain on phenol. Furthermore,

a series of strains harbouring plasmids lacking expres-

sion of each of the genes in turn confirmed that all six

of these genes are required (Nordlund et al. 1990a).

However, these results did not rule out the possibility

that one or more of these gene products is involved in

something other than hydroxylase activity, for example

phenol transport.

At the time the nucleotide sequences of these genes

were determined, database searches revealed similari-

ty only for the amino terminal of P5 (38.5 kDa) with

other proteins. These are plant-type ferredoxins, which

are small (11 kDa) electron-transfer proteins contain-

ing an iron-sulfur centre that is liganded by four cys-

teine residues with characteristically well-conserved

spacing. Purification of P5 revealed the presence of

flavin adenine dinucleotide (FAD) in addition to the

expected [2Fe-2S] centre, which was confirmed by

visible absorbance spectroscopy to be of the ferredox-

in type (Powlowski & Shingler 1990). The purified

protein catalyzes the NAD(P)H-dependent reduction

of cytochrome c, a property that is shared with other

proteins containing these prosthetic groups (reviewed

in Mason & Cammack 1992). The role of the fiavin in

these reductases is to accept electrons from the obligate

2-electron donor, NAD(P)H, and presumably to then

pass them one at a time to the oxygenase active site on

another polypeptide via the [2Fe-2S] centre. In some

multicomponent oxygenases the ravin and iron-sulfur

centre prosthetic groups may be present on separate

polypeptides (Fig. 3).

Since this work was published, sequences for a

number of these electron-transferring proteins have

appeared, and up-to-date comparisons including P5

can be found in another review in this volume

(Williams & Sayers 1994). Possible evolutionary rela-

tionships between ferredoxin-NADP + reductases and

P5, as well as other oxygenase reductases, are dis-

cussed by Andrews et al. 1992.

Further characterization of phenol hydroxylase,

and the roles of the five other polypeptides, P0-P4,

required the development of an in vitro assay and purifi-

cation of the individual components. In vitro activity of

the enzyme was found to be dependent on the addition

of NADH, or, less effectively, NADPH, to an assay

mixture, in addition to Fe +2 (with Fe +3 stimulating

a lower level of activity). 1 The polypeptides required

for hydroxylase activity were identified by assaying

crude extracts from strains in which genes for each

polypeptide in turn had been deleted. These experi-

ments revealed that of the six polypeptides demon-

strated to be required for growth on phenol by a

catechol-degrading test strain, only P0 was dispens-

able for hydroxylase activity ~owlowski & Shingler

t990). The function of PO, whose primary structure

does not resemble that of any protein currently in the

data bases, z is not yet known but roles in phenol trans-

port or hydroxylase regulation are two possibilities.

Purification of P1-P4 (J Powlowski & V Shingler,

unpubl.) resolves these polypeptides into two com-

ponents, one consisting of P2 alone, and the other a

complex of P1-P3-P4 (see Fig. 2). The purified pro-

teins are active in the in vitro assay when supplemented

with the reductase (P5) (Powlowski & Shingler 1990,

unpubl.). Visible absorbance spectroscopy showed that

none of the purified P1-P4 components appear to con-

tain any of the prosthetic groups commonly associated

with microbial aromatic mono- and di-oxygenases (see

Fig. 3). However, Fe +2 is still required in the assay,

raising the possibility that one of the components had

lost an intact iron centre: since S -2 was not added to

the assay, reconstitution of an iron-sulfur centre is not a

likely explanation. The presence of a Reiske-type iron-

sulfur centre, common in numerous oxygenase com-

ponents (Fig. 3), is also unlikely, based on sequence

comparisons with other Reiske-centre containing pro-

teins (Powlowski & Shingler 1990).

On the basis of these studies a general similarity

between the phenol hydroxylase from this organism

and methane monooxygenase was noted (Powlowski

& Shingler 1990). Thus, both enzymes are made up of

five polypeptides of similar sizes: one is a reductase

with FAD and a ferredoxin-type [2Fe-2S] centre; three

co-purify, and in the case of methane monooxygenase

have been demonstrated to comprise the oxygenase

itself; and one is a low molecular-weight polypeptide

that can be resolved from the others and which possess-

es no chromophoric prosthetic group (P2 in phenol

hydroxylase, component B in methane monooxyge-

nase) (see Fig. 3). The function of the latter component

in methane monooxygenase is somewhat controversial,

but appears to be involved in regulation of the oxyge-

nase activity (Green & Dalton 1985; Froland et al.

1992). The sequence similarities between components

of these two oxygenases include 28% identity between

the reductases of the two systems (Stainthorpe et al.

1989, 1990; Nordlund et al. 1990a). The next high-

est sequence similarity is found between the P2 and

MmoB components of the two systems, which share

27% identity if two large gaps are included to optimise

alignment. However, little overall sequence identity

is evident between the other polypeptides of phenol

hydroxylase and methane monooxygenase, even after

inclusion of multiple gaps.

Among the properties not yet known about phenol

hydroxylase is the identity of the oxygen-activating

prosthetic group. One possible candidate, despite the

lack of overall sequence identity, is the binuclear iron

centre like that of methane monooxygenase (Fox et

al. 1988; Ericson et al. 1988; Rosenzweig et al.

1993). While variants of this centre axe found in a

number of different enzymes, that found in the e~-

subunit of the A-component of methane monooxyge-

nase (MmoX) is similar to the binuclear iron centre of

ribonucleotide reductase, to which methane monooxy-

genase appears to be very distantly related (Nordlund

et al. I992). The crystal structure of ribonucleotide

reductase revealed that the binuclear iron centre is lig-

anded by two histidine, one aspartate, and three glu-

tamate residues (Nordlund et al. 1990b), while the

crystal structure of methane monooxygenase showed

Iigation by two histidine and four glutamate residues

(Rosenzweig et al. 1993). The spacing of these ligands

is conserved in methane monooxygenase and ribonu-

cleotide reductase, as well as in the P3 potypeptide of

phenol hydroxylase (Fig. 4). Despite the lack of over-

225

all sequence identity between ribonucleotide reductase

and methane monooxygenase, the active site struc-

tures of the two enzymes are quite similar (Nordlund

et al. 1992; Rosenzweig et al. 1993). Therefore the

tack of strong sequence identity around the putative

binuclear iron centre ligands of P3 is not particular-

ly worrisome. It is interesting to note that Thr-213 of

methane monooxygenase, thought to play an important

role in some dioxygen-activating centers is conserved

as Thr-204 in P3 (Fig. 4; Rosenzweig et al. 1993, and

references therein).

Several polypeptides of phenol hydroxylase also

share low-level sequence homology with polypeptides

of toluene-4-monooxygenase from Pseudomonas men-

docina KR1. The sequence similarities between these

two proteins have been noted previously and include

homologies with the P1, P2, P3 and P5 polypeptides

of phenol hydroxylase (Yen et al. 1991; Yen & Karl

1992). It is interesting to note that putative binuclear

iron centre ligands are also found in TmoA of toluene

monooxygenase, which shares 35% overall sequence

identity with P3 (Fig. 4). Although the presence of a

binuclear iron centre in toluene and phenol hydroxy-

lases must be confirmed by biochemical studies of the

purified proteins, it appears that oxygenases contain-

ing this centre are not limited to methane monooxyge-

nase.

Phenol hydroxylases from other bacterial strains

The oxygenation of electron-rich phenol is a consid-

erably easier task than the oxygenation of toluene

or methane. Considering that a single FAD-binding

polypeptide is so commonly used by microbes for

hydroxylation of phenolics, it is at the moment puz-

zling that Pseudomonas sp. strain CF600 elaborates

such a complex phenol hydroxylase. Indeed, two other

species of Pseudomonas, P. pickettii PKO1 and Pseu-

domonas EST1001, have clearly been shown to utilize

phenol hydroxylases that are single-component flavo-

proteins related to other aromatic flavoprotein hydrox-

ylases (Kukor & Olsen 1992; Nurk et al. 1991). Further

study of the multicomponent hydroxylase might hint

at what advantages are conferred to compensate for

the energetic expense of synthesizing six polypeptides

rather than one.

These considerations prompted us to ask whether

the multicomponent phenol hydroxylase is an orphan

or whether other phenol degrading bacteria also use

related multicomponent phenol hydroxylases. Reports

in the literature indicated that not all bacterial phenol

226

EcRNR

DmpN(P3}

MmoX

TmoA

P~EKHIFISNL~QTL~QGRSPNVALLPLIS

DARYVNAL_KLFLTAVSPL [E[YQAFQGFSRVGRQFSG

HPKWNE TMKVVSNFLEVG I E [ YNAIAATGMLWD SAQA

DPGWISTL_KSNYGAIAVG E~ ZA~_--VTGEGRNARFSKA

I PELE TWVE TWAFS TI SRSYTH I II~d~IVN- 132

-AGARVACQMQAI DIE[ LR IH[ VQTQVHAMSIIYNK- 155

-AEQKNGYLAQVLD_-]E] I~ [H I THQCAYVNYYFAK-160

-PGNRNMATFGMMD EL~ LR [HI GQLQLFFPNEYC_K-150

EcRNR

DmpN(P3)

MraoX

TmoA

LRELKKKLYLCLMSVNAL

]E I

AI RFYVSFACS FAFAERELME GNAKI IRLIARD_ LTGTQHMLNLLRS-255

DARTAGPFE FLTAVSFSF [El YVLTNLLFVPFMSGAAYNGDMATVTFGFS_AQSD MTLGLEVIKFMLE-250

GFI SGDAVECSLNLQLVG I E [ ACFTNPLIVAVTEWAAANGDE I TPTVFLS iErD ~ S~ ~a MANGYQTVVS IAN-259

I I TGRDAI SVAIMLTFSF LE j TGFT-NMQ FLGLAAD~EAG-Dy T FAN L I SS-~Q T5 _-- AQQGG PALQLL I E - 247

Fig.4.

Conservation ofligands for the binucleariron centre ofdifferent enzymes. The consensus sequenceand extent ofhelices ofribonucleofide

reductase are taken from Nordlund et al. (1990b). E.

coti

ribonucleotide reductase (EcRNR, Carlson et aL 1984), DmpN (P3, Nordlund et al.

1990a), MmoX (Stainthorpe et al. 1990; Rosenzweig et al. 1993), TmoA (Yen et al. 1991). Ligating residues are boxed, other residues present

in at least three out of the four sequences shown are underlined.

hydroxylases are simple flavoproteins (Gurujeyalaksh-

mi & Oriel 1989), and that some multicomponent oxy-

genases for compounds like toluene are capable of turn-

ing over phenols (Spain & Gibson 1988). Considering

the surprising complexity of the phenol hydroxylase

revealed by our studies, it seemed unlikely that similar

enzymes would readily have been isolated, especially

if a single-component enzyme was expected. In order

to address this question, DNA from a collection of

phenol-degrading bacteria was probed for the presence

of each of the phenol hydroxylase genes.

Gene-probing experiments were performed on

eleven phenol-utilizing bacteria in addition to

Pseu-

domonas

sp. strain CF600. One of these strains was R

putida

U, the archetypal phenol-degrader whose phe-

no1 hydroxylase has not been characterised. The other

ten were marine strains collected off the Norwegian

coast. Southern hybridization analysis demonstrated

that five of the marine isolates as well as

R putida

U all possess DNA highly homologous to each of the

components of the

dmp-encoded

phenol hydroxylase.

Furthermore, all of the strains that tested positive were

also found to posses DNA highly homologous to all

nine

meta-cleavage

pathway genes of the

dmp-operon,

and to the specific regulator of phenol catabolism of

this strain (Nordlund et al. 1993).

The nucleotide sequence of the chromosomally-

encoded phenol hydroxylase region from another

phenol-degrading pseudomonad,

P. putida

P35X, has

also recently been determined (Ng et al. 1993). This

strain, like

Pseudomonas

sp. strain CF600, utilizes

a multicomponent phenol hydroxylase and a

meta-

cleavage pathway. The deduced amino acid sequences

from the six open reading frames of the phenol hydrox-

ylase region share between 89% and 99.7% identity

with those of P0 to P5 (LC Ng, pers. comm.).

It is interesting to note that all of these organ-

isms catabolize phenol using a

meta-cleavage

path-

way, while marine isolates that degrade phenol via

ortho-cleavage

pathway lack DNA homologous to

the multicomponent phenol hydroxylase. However,

this arrangement does not always hold, as a single-

component flavoprotein phenol hydroxylase has been

shown to be associated with the

meta-cleavage

pathway

P. pickettii

PKO1 (Kukor & Olsen 1991). Neverthe-

less, it is now clear that the multicomponent phenol

hydroxylase is not unique to

Pseudomonas

sp. strain

CF600. In all likelihood related enzymes will turn out

to make up a rather significant fraction of the hydrox-

ylases used in aromatic metabolism.

The meta-cleavage pathway for catechol

The biochemical route of the

meta-cleavage

pathway

following phenol hydroxylation is illustrated in Fig. 1,

and the function of the

drop-encoded

enzymes involved

are summarised in Table 1. With the exception of iso-

functional genes homologous with

dmpB and dmpQ,

no other

meta-cleavage

enzyme gene sequences had

been published prior to work with the

dmp

system.

Furthermore, only the amino acid sequence of DmpC

was similar enough to other sequences in the databas-

es to deduce a function with any confidence. Hence

assignment of the functions of individual genes relied

almost exclusively on correlating enzymatic activi-

ty with expression of individual genes. Although the

majority of the enzyme activities of the

meta-cleavage

pathway had been discovered by the mid-seventies, this

type of analysis of the

dmp

operon led to the discovery

of a new meta-cleavage pathway enzyme, aldehyde

dehydrogenase (acylating) (ADA, see Fig. 1, Table

1). In the following sections we will review the indi-

vidual enzymes of the pathway, and where appro-

priate, or where evidence is lacking for the

dmp-

encoded enzymes, isofunctional enzymes of other

meta-cleavage

operons. In particular, reference will

be made to the four

meta-cleavage

pathway systems

shown in Table 2, for which the sequences of all

meta-

cleavage pathway genes are known.

Catechol-2,3-dioxygenase: Cleavage of the aromatic

ring

This enzyme catalyzes the critical ring-opening step of

the

mere-cleavage

pathway (Fig. 1), and contains non-

heine Fe +2 at the active site (reviewed by Yamamo-

to & Ishimura 1991). The

dmpB-encoded

enzyme

shares 83-87% sequence identity with catechol 2,3-

dioxygenases from other

meta-cleavage

pathways of

different

Pseudomonas

species (Bartilson & Shingler

1989). Much lower levels of sequence similarity are

shared with other meta-cleavage dioxygenases (Table

2) that form a different evolutionary subgroup. Evo-

lutionary relationships, and up-to-date comparisons of

different ring cleavage 2,3-dioxygenases are reviewed

in this volume by Williams & Sayers (1994). Despite

the fact that catechol-2,3-dioxygenase was the first

meta-cleavage

pathway enzyme to be isolated and

crystallized, no structure or detailed mechanism has

yet been elucidated for any catechol-2,3-dioxygenase.

However, some progress has been made in manipu-

lating the substrate specificities of the xylE-encoded

catechol 2,3-dioxygenase using genetically-selected

mutants and chimeric proteins (Wasserfallen et al.

t991; Williams et al. 1990).

DmpQ: A protein with homology to plant-type

ferredoxin

The

dmpQ

gene, located between the genes encod-

ing phenol hydroxylase and catechol-2,3-dioxygenase,

encodes a polypeptide about the size of, and with up

to 41% sequence identity with, plant-type ferredoxins

(Shingler et al. i992). Homologous proteins, encod-

ed in analogous locations of the xyl and

nah

operons,

227

share 64% and 52% identity with DmpQ (Harayama

& Rekik 1993; Harayama etal. 1991; You et al. 1991).

Studies with strains in which

dmpQ

has been deleted

from the operon indicate that expression of this protein

is required to allow strains to grow at the expense of 4-

methylphenol or 3,4-dimethylphenol, but not phenol,

2-methylphenol or 3-methylphenol (V Shingler & J

Powlowski unpubl.).

This growth pattern is reminiscent of that recent-

ly reported for mutants of the xyl homologue (xylT),

namely lack of growth on 4-methyl substituted sub-

strates. The xyl homologue has been shown, using

vivo

studies, to be involved in reactivation of catechol-

2,3-dioxygenase (Polissi & Harayama 1993). This

enzyme is particularly sensitive to inactivation dur-

ing turnover with 4-substituted catechols, probably by

inadvertent oxidation of the active-site ferrous iron.

Reactivation is postulated to involve transfer of elec-

trons from an as-yet unidentified donor through XylT

to the oxidized iron. Considering the high degree

of similarity between the

dmp

and xyl operons, and

the qualitatively similar growth patterns of

dmpQ and

xylT

mutants, it is likely that DmpQ performs the

same function as XylT. It is interesting to note, that

while the sequence identity between the catechol 2,3-

dioxygenases in the two strains is 84%, XylT and

DmpQ share only 64% identity. It is possible that these

ferredoxins have evolved to accommodate interaction

with different electron donors in the two strains. Hence

the greater divergence of XylT and DmpQ might reflect

these accommodations rather than a response to struc-

tural differences between the corresponding catechol-

2,3-dioxygenases with which they presumably inter-

act.

The presence of the ferredoxin-like protein in the

xyl-, dmp-

and

nah-encoded

pathways supports the

idea that inclusion of these redox proteins represents

a bacterial strategy to expand the substrate speci-

ficity of the

meta-cleavage

pathway to include com-

pounds chanelled through the otherwise suicidal 4-

methylcatechol (Polissi & Harayama 1993). In this

respect the observation that neither the

tod-

nor the

bph-

abridged

recta-pathway

operon appears to encode a

similar protein is illuminating (Lau et al. 1993; Kikuchi

eta|. 1993). In both of these enzyme systems, no com-

pounds that would be metabolised

viapara-substituted

intermediates have been shown to be substrates of the

respective pathways.

228

2-Hydroxymuconic semialdehyde dehydtvgenase

(HMSD) and hydrolase (HMSH): gatekeepers at the

pathway branch point

These enzymes both use ring cleavage products as

substrates, which are 2-hydroxymuconic semialdehyde

from catechol, 5-methyl-2-hydroxymuconic semialde-

hyde from 4-methylcatechol, and 2-hydroxy-6-oxo-

2,4-heptadienoate from 3-methylcatechol. Since the

ring-cleavage product of 3-methylcatechot is a ketone,

rather than an aldehyde, it cannot be further oxidized by

the dehydrogenase, and must therefore be metabolised

via the hydrolytic route (Fig. 1). This was originally

shown using hydrolase-defective strains of

R putida U

that failed to grow at the expense of phenols that are

channelled via the

meta-cleavage

pathway through 3-

methylcatechol (Bayly & Wigmore 1973). Despite the

potential for use of either branch for the ring-cleavage

products of catechol or 4-methylcatechol, these com-

pounds were shown to be preferred substrates for the

dehydrogenase, rather than the hydrolase of the

meta-

cleavage pathway of

P. putida

U (Sala-Trepat et al.

1972). Conclusions from these in vitro results were

reinforced by the observation that mutants of

P. puti-

U defective only in the 4-oxalocrotonate branch

failed to grow at the expense of phenols channelled

through catechol or 4-methylcatechol (Bayly & Wig-

more 1973).

These conclusions are also apparently valid for the

dmp-encoded meta-cleavage

pathway. Thus, a dele-

tion within the HMS-dehydrogenase gene

(dmpC),

either of the genes for the other two enzymes of the

4-oxalocrotonate branch

(dmpl

and

dmpH),

resulted

in strains that grew on 2-methyl, 3-methyl and 3,4-

dimethylphenols, but not on phenol or 4-methylphenol.

On the other hand, strains deleted within

dmpD,

the

gene encoding the HMS-hydrolase, grew on phenol

and 4-methylphenol, but not at the expense of the oth-

er substrates (V Shingler & J Powlowski unpubl.).

Despite the structural similarity of the substrates

of both enzymes, the lack of sequence homolo-

gy between HMS-hydrolase and HMS-dehydrogenase

indicates different evolutionary origins. The amino

acid sequence of HMS-dehydrogenase shows approxi-

mately 40% identity with various eukaryotic aldehyde

dehydrogenases (Nordlund & Shingler, 1990), and

84% identity with xyl-encoded HMS-dehydrogenase

(Horn et at. 1991). Speculations about the possi-

ble evolutionary significance of this similarity have

been presented for the xylG-encoded enzyme (Horn

et al. 1991), and can equally well be applied to

the dmpC-encoded

counterpart. Sequence comparison

searches for HMS-hydrolase, on the other hand, only

readily identify isofunctional hydrolases including the

xyl and

tod-encoded

enzymes, as well as hydrolases

for phenyl-substituted 2-hydroxymuconic semialde-

hyde, which is an intermediate in microbial biphenyl

metabolism (Table 2). In the latter case, the sequence

identity is considerably lower than that for the oth-

er HMS-hydrolases, possibly reflecting differences in

substituent bulkiness on the substrate.

While information about structure-function rela-

tionships for any HMS-hydrolase or HMS-dehydro-

genase is non-existent, the sequence comparisons sug-

gest avenues for further experimentation. Consider first

the striking similarity between HMS-dehydrogenase

and eukaryotic aldehyde dehydrogenases. While

chemical modification studies have implicated a num-

ber of residues in enzyme activity, including Cys-

302 and Glu-268 of horse aldehyde dehydrogenase

(conserved as Cys-288 and Glu-254 in

dmp-encoded

HMS-dehydrogenase), assignment of definite roles for

these residues has been difficult (reviewed in Hempel

& Jrrnvall 1989). By analogy with glyceraldehyde-3-

phosphate dehydrogenase, the mechanism of aldehyde

dehydrogenase has often been assumed to involve for-

mation of a thiohemiacetal enzyme-substrate interme-

diate that transfers a hydride equivalent to NAD +, and

is then hydrolyzed from the enzyme. However, com-

pelling evidence has been presented that an active-site

serine residue of sheep-liver aldehyde dehydrogenase

is invoh, ed in formation of an acyl-enzyme intermedi-

ate (Loomes et at. 1990). It is interesting that the serine

residue implicated in these studies is conserved in alde-

hyde dehydrogenase isozymes (Johansson et al. 1988),

while it is not conserved in the HMS-dehydrogenase

sequence. HMS-dehydrogenase thus provides a new

variation with which to test roles proposed for the var-

ious conserved aldehyde dehydrogenase residues, and

to probe the involvement of an active-site serine residue

in catalysis.

The participation of an active site serine residue

in HMS-hydrolase catalysis has also been suggested

on the basis of low sequence identity (20% overall)

of the isofunctional xy/-encoded enzyme with atropine

esterase, a serine hydrolase (Horn et al. 1991). This

observation can be extended to note that Set-107 of

the

dmp-encoded

HMS-hydrolase lies within an active-

site lipase consensus sequence GXSXG (reviewed by

Derewenda & Sharp 1993). HMS-hydrolase shares

24% overall identity with a lipase from

Moraxella

(Feller et al. 1991). The serine residue within the lipase

229

consensus sequence is coupled in lipases to Asp (or

Glu) and His, much like the archetypal catalytic triad

of serine proteases (see Derewenda & Sharp 1993).

On the basis of these comparisons it is conceivable

that the mechanism of HMS-hydrolase, which cleaves

a carbon-carbon bond, is similar to the mechanisms of

esterases, in which a carbon-oxygen bond is cleaved. It

is interesting to note that the lipase consensus sequence

with a Cys replacing Ser is a motif conserved in the

ortho-cleavage pathway enzyme dienelactone hydro-

lase (Pathak & Ollis 1990).

4-Oxalocrotonate isomerase (401): a small but

essential participant in the HMS-dehydrogenase

initiated branch

4-oxalocrotonate isomerase (401) catalyzes the iso-

merization of 4-oxalocrotonate, the product of HMS-

dehydrogenase, to 2-keto-3-hexenedioate (Fig. 1).

Studies using mutants of

P. putida U lacking 4OI activ-

ity showed that this enzyme is necessary to support

growth on phenols channelled through catechol or 4-

methylcatechot, which are metabolized via this branch

(Wigmore et al. 1974). As indicated earlier, this is also

the case for the dmp-encoded pathway, since a dele-

tion within the 4OI-encoding gene (dmpl) of the oper-

on prevented the resulting strain from growing at the

expense of phenol and 4-methylphenol. These results

demonstrate the indispensability of 4OI, which is sig-

nificant since the non-enzymatic isomerization reac-

tion is rather fast and could have been sufficient to

support growth in the absence of a specific enzyme

(Sala-Trepat & Evans 1971).

The sequence of 4OI shares 78% identity with

the isofunctional xylH-encoded enzyme, but no sim-

ilarity is found with other protein sequences. 4-

Oxalocrotonate isomerase encoded by the xyl operon

has been isolated and partially characterized (Chen et

al. 1992). Mechanistic studies suggest that the isomer-

ization of

2-oxo-4-trans-hexenedioate proceeds via the

enol form of the substrate shown in Fig. 1, and in

this respect at least, 4OI resembles 3-oxo-AS-steroid

isomerase, as well as other isomerases (Bayly & Bar-

bour 1984; Whitman et al. 1991). It will be interest-

ing to compare the properties of these two isofunc-

tional 4Ois, as 22% of the amino acid sequence is

not identical, in order to see what conclusions can

be drawn about structure-function relationships. X-ray

structures of both the x3'l-encoded enzyme (Davenport

& Whitman 1993) and the drop-encoded enzyme (D.

Roper unpubl.) are currently being sought.

4-Oxalocrotonate decarboxylase (,COD) and

2-oxopent-4-dienoate hydratase (OEH): A complex of

related polypeptides

4OD catalyzes the final step in the 4-oxalocrotonate

branch, while OEH is involved in the subsequent

reaction, which occurs after the 4-oxalocrotonate and

hydrolytic branches merge (Fig. 1). The first attempt-

ed purification of OEH, from phenol-grown P. putida

U, was reported by Collinsworth et al. (1972). The

enzyme proved difficult to purify, and could not be

resolved from some persistent contaminants. One of

the contaminants was undoubtedly 4OD, as years later

it was shown that xyl operon-encoded 4OD and OEH

were tightly enough associated with each other to co-

purify. As the unstable product of 4OD is the substrate

for OEH, it has been proposed that the physical associa-

tion between the two enzymes has arisen to channel the

unstable intermediate (Harayama et al. 1989). Howev-

er, as these workers pointed out, HMS-hydrolase of

the hydrolytic branch generates the same intermedi-

ate, and this enzyme is not tightly associated with the

hydratase (OEH) in the x3,l-encoded system. A loose

association that still allows metabolite channelling has

not been ruled out.

Sequencing of the genes for OEH and 4OD from the

dmpoperon suggested a possible evolutionary expla-

nation for the association of these two polypeptides

(Shingler et al. 1992). As is the case for the xyl-

encoded homologues, the dmp-encoded enzymes are

also tightly associated with each other, and co-purify

(J. Powlowski unpubl., but see Fig. 2). When opti-

mally aligned, their deduced amino acid sequences

show 37% identity, indicating common ancestry. It is

therefore possible that these enzymes evolved from a

protein that was originally multimeric, and this may be

why they are so tightly associated with each other. The

importance of channelling, which could result from

such an arrangement, remains to be demonstrated. If

metabolite channelling is indeed important it may have

played a role in maintaining selective pressure on the

multimeric form of the enzymes.

It seems rather remarkable that an enzyme that cat-

alyzes carbon-carbon bond cleavage is related to one

that involves hydration of a double bond. However,

similar electron shifts can reasonably be postulated for

these two reactions (Fig. 5A, Dagley 1975), so evo-

lution of the two enzymes from a common ancestor

would perhaps not be too surprising. In both cases,

the keto group of the substrate could act as an elec-

tron sink, especially when complexed with a metal

230

DmpH DrapE

coon ~3 .coon

COOH CH3 COOH

O= HO/~ ~ "

DmpG Ni_fV

COOH

~3 coon no.I o

COOH

+ .COOH

H=c I ~co A

Fig. 5.

Comparisons of reactions catalyzed by DmpE (OEH) and

DmpH (4OD) (A), and DmpG (HOA) and NifV (B). While the

mechanisms of these enzymes are as yet not known, these reasonable

possibilities are illustrated for comparative purposes (see text).

ion, a requirement for which has been demonstrat-

ed for both the decarboxylase (4OD, Sala-Trepat &

Evans 1971; Harayama et al. 1989) and the hydratase

(OEH, Collinsworth et al. 1972; Harayama et al. 1989).

However, it is not currently known whether the iso-

mer of the hydratase substrate shown in Fig. 5A is

catalytically competent. 2-Keto-4-pentenoate, which

is the form shown in Fig. 1, is in rapid equilibri-

um with the enol form, 2-hydroxy-2,4-pentadienoate

(Marcotte & Walsh 1978), which is competent for

turnover by OEH (Harayama et al. 1989). A solution

of these isomers decays to trans-2-keto-3-pentenoate

(Marcotte & Walsh 1978) which is probably the form

not metabolised by the enzyme (Harayama et al. 1989).

The involvement of cis-2-keto-3-pentenoate (the iso-

mer shown in Fig. 5A), as a substrate or transient inter-

mediate in the OEH catalyzed reaction, has not been

specifically addressed.

It is worth emphasizing that from comparisons of

the sequences of all meta-pathway proteins, dmpE

(hydratase, OEH) and dmpH (decarboxylase, 4OD)

represent the only example of possible evolution by

gene duplication. None of the other protein sequences

show significant similarity with each other. Hence,

gene duplication and divergence were not foremost

in evolution of this pathway (Shingler et al. 1992). In

this respect the comparatively low homology between

the hydratase (OEH)-encoding genes of the dmp-

encoded meta-pathway arid those of the abridged tod-

and bph-encoded meta-pathways is notable (Table 2).

Metabolism of the substrates of the tod and bph-

systems does not require the 4-oxalocrotonate branch

of the pathway, and in neither case do the respec-

tive operons include genes for this branch (Lau et al.

1993; Kikuchi et al. 1993, pers. comm.). Therefore,

in these abridged meta-pathways the hydratase coun-

terparts, which use exactly the same substrate as the

dmp-encoded enzyme, would have evolved under dif-

ferent conditions i.e., in the absence of a physically-

associated decarboxylase partner.

4-Hydroxy-2-ketovalerate aldolase (HOA) and

aldehyde dehydrogenase (acylating) (ADA): A second

example of physical association and a new

meta-cleavage pathway enzyme

Close physical association has also been conclu-

sively demonstrated for the last two enzymes of

the dmp-encoded pathway, 4-hydroxy-2-ketovalerate

aldolase (HOA) and aldehyde dehydrogenase (acylat-

ing) (ADA), which generate the end-products pyruvate

and acetyl-CoA, respectively. Like the two preceed-

ing enzymes, HOA and ADA purify to homogeneity

(Powlowski et al. 1993, Fig. 2), but unlike them do not

share common ancestry. Close association in this case

may be required to channel the aldehyde produced by

the aldolase either to reduce toxicity or to ensure that

it is metabolized by ADA, and not some less efficient

route (see below). A consequence of this association

appears to be that the activity of HOA is regulated

by NAD +, and to a lesser extent NADH, which are

used not by the aldolase itself, but by its partner, ADA

(Powlowski et al. 1993). The mechanism by which this

occurs has not yet been elucidated, but conceivably

involves allosteric interactions. This phenomenon, and

the possibility of channelling, may be significant for

modulation of pathway activity at the enzyme level.

Aldolases in general can be divided into two class-

es: those that require a metal ion, and those that rely

on an active-site lysine, for interaction with the keto-

group of the substrate to provide an efficient electron

sink (Walsh 1979). It has long been known that the

meta-cleavage

aldolase, HOA, requires a metal ion

for activity. While that from P.

putida

U is apparent-

ly stimulated by Mg +2 (Dagley & Gibson 1965), the

drop-encoded

HOA is stimulated by Mn +2, with no

increase in activity observed using Mg +2 (Powlowski

et al. 1993). However, the enzyme is still active in the

absence of added metal ions, and no attempt has yet

been made to correlate metal content of the enzyme

with activity.

The

drop-encoded

HOA shares low sequence

homology with enzymes that catalyze reactions that are

mechanistically the reverse of the aldol cleavage reac-

tion catalyzed by HOA (Shingler et al. 1992, and refer-

ences therein). One example is NifV, which is thought

to encode homocitrate synthase that catalyzes Claisen

condensation of acetyl-CoA and o~-ketoglutarate (Fig.

5B).

For years the meta-cleavage pathway has been

depicted as ending at pyruvate and acetaldehyde (or

propionaldehyde in the case of 4-methyl-substituted

growth substrates). Work with the

drop

operon has

uncovered the existence of a new

rneta-cleavage

oper-

on encoded enzyme that converts the aldehyde to the

acyl-CoA derivative using NAD + and coenzyme A

(ADA; Shingler et al. 1992; Powlowski et al. 1993).

Metabolism in other organisms involves oxidation of

acetaldehyde to acetate, followed by ATP-dependent

conversion to the CoA ester (reviewed in Nunn 1987).

The ATP-independent reaction catalyzed by ADA thus

represents an energetically efficient mechanism for

catabolism of the short-chain aldehyde formed by the

pathway. However,

Pseudomonas

CF600 grown at the

expense of phenol also has low levels of enzymes

involved in an ATP-dependent pathway, so some

metabolism of acetaldehyde or propionaldehyde by

these enzymes is possible. It is difficult to test direct-

ly whether or not the

drop-enzyme

is absolutely nec-

essary, as the close coupling of ADA activity with

HOA activity means that mutations or deletions within

the ADA-gene also affect HOA activity second-hand

(Shingler et al. 1992).

Apart from recently-discovered isofuncfional

enzymes (Table 2), the sequence of aldehyde dehy-

drogenase (acylating) (ADA) is not similar to any oth-

er proteins so far entered into the databases. Howev-

er, the amino-terminal of the protein may encompass

the NAD+-binding site, as it contains a characteris-

tic ADP-binding fingerprint sequence (Wierenga et al.

1986, Shingler et al. 1992).

Sequence information is available for at least

one other bacterial CoA-dependent dehydrogenase,

231

namely methylmalonate semialdehyde dehydrogenase

(Steele et al. 1992), and its rat counterpart (Kedishvili

et al. 1992). It is interesting that these sequences show

higher similarity to the CoA-independent eukaryot-

ic aldehyde dehydrogenase, than to the

dmp-encoded

CoA-dependent dehydrogenase, ADA. This suggests

at least two separate evolutionary origins for the

CoA-dependent dehydrogenases, possibly involving

one ancestral NAD +-binding protein and another that

bound CoA. It is perhaps even more intriguing that the

CoA-independent HMS-dehydrogenase from the

dmp-

encoded pathway is also related to eukaryotic alde-

hyde dehydrogenases but not to the CoA-dependent

dehydrogenase (ADA). When the two

dmp-encoded

dehydrogenases are directly compared they show <

20% identity even with insertion of sixteen gaps to

optimise the alignment. Thus, while these two dehy-

drogenases of the

meta-cleavage

pathway might have

been predicted to have evolved from a common ances-

tor, sequence comparisons indicate they have not. It

will be interesting to compare the sequences of other

biochemically-characterized bacterial CoA-dependent

dehydrogenases, e.g.,

Clostridium

acetaldehyde dehy-

drogenase (acylating) (Burton & Stadman 1953), when

they become available.

Transcriptional regulation of the dmp operon

Transcription of the

dmp

operon is tightly regulat-

ed by the divergently transcribed

dmpR

gene prod-

uct (see Fig. 1). As with other regulators of aromatic

catabolism, the activity of the regulator is itself modu-

lated by the presence of aromatic compounds to allow

expression of the specialized catabolic enzymes only

when the substrates of the pathway are present (Shin-

gler et al. 1993). In this capacity the aromatic com-

pound serves as an effector molecule. For a given com-

pound to support growth as the sole carbon and energy

source, it must therefore serve as both an effector for

the regulator and as a substrate for the enzymes of the

pathway. Hence, the effector recognition specificity of

the regulator, in addition to specificities of the catabol-

ic enzymes, is intimately involved in determining the

range of compounds that can be degraded by a metabol-

ic pathway. This has been most elegantly exploited

in manipulation of the pWW0-encoded XylS regula-

tor of benzoate metabolism. Studies of this regulator

have shown that new substrates for thepWW0-encoded

pathway could be selected by mutational expansion of

the effector specificity range of XylS (Abril et al. 1989;

Ramos et al. 1986, 1990).

232

DmpR, like the pWW0-encoded XylR regulator of

toluene and xylene catabolism, belongs to the exten-

sive NtrC-family of transcriptional activators (Shin-

gler et al. 1993). Members of this family respond

to diverse environmental signals to regulate genes

involved in a variety of physiological processes. These

transcriptional activators act by binding to enhancer-

like elements, and regulating transcription from a dis-

tinct set of promoters recognised by ~r54-holoenzyme

RNA polymerase (reviewed in North et al. 1993). As

with eukaryotic enhancer-binding proteins, members

of the bacterial NtrC family are composed of distinct

functional domains. The central and carboxy-terminal

domains of these activators are conserved among all

members of the family and are believed to be involved

in interaction with the 0 .54 RNA polymerase and in

DNA binding, respectively. In many members of the

family the amino-terminal A domains are the sites of

transfer of the signals that are received via sensory

proteins. However, the A domain of DmpR shares

64% homology with the equivalent domain of XylR,

but does not share homology with other members of

the NtrC family or other proteins in the data bases

(Shingler et al. 1993). The unique A-domain homol-

ogy shared by DmpR and XylR, in conjunction with

the signal receptor function of the A-domains in other

members of the family, suggested that this region is

involved in the activation of DmpR and XylR by their

respective aromatic effectors. Studies of the response

of hybrid DmpR/XylR regulators, in which all or parts

of the A-domain of DmpR were exchanged with that of

XylR, have now clearly shown that the distinct effec-

tot recognition specificities of the two regulators spans

and completely resides within the amino terminal 234

residues (Shingler & Moore 1994).

Both DmpR and XylR have broad effector speci-

ficity, but respond differentially depending on the posi-

tion and nature of the substituent(s) on the aromatic

ring of the effector (Abril et al. 1989; Shingler &

Moore 1994). In the case of DmpR the response to

phenolic compounds substituted in the

para-position

relatively poor, suggesting that the inefficient growth

observed on these compounds might be due to low

expression of the catabolic enzymes. This is apparent-

ly the case, since a

Pseudomonas

strain carrying the

dmp

system on an RSF1010-based ptasmid of 16-20

copies/celt has a generation time of 66 minutes in min-

imal media containing 4-methylphenol as the carbon

source. This compares favourably with the 132 minute

generation time of this strain harbouring the 1-2 copy

number pVI 150 wild-type plasmid (H. Pavel & V. Shin-

gler unpubl.). However, the improved 4-methytphenol

degrader was unable to grow on phenol, a phenomenon

associated with hyperproduction of the phenol hydrox-

ylase in response to the efficient effector property of

phenol. Although not specifically tested, growth inhi-

bition in this case may be due to interference with

aromatic amino acids, a possibility suggested by the

observation that expression of phenol hydroxylase in

E. coli

results in accumulation of a brown mixture of

pigments produced from aromatic amino acids in the

media &owlowski & Shingler 1990). The detrimen-

tal effects of wholesale over-expression in this case

suggests that more subtle modifications, such as effec-

tor specificity mutants, are likely to be more success-

ful in construction of strains with all-round improved

degradative properties.

Most aromatic catabolic regulators so far analysed

are members of one of three families of bacterial tran-

scriptional regulators: the LysR family e.g., NahR,

CatM, CatR, TcbR, TfdS and ClcR (see Coco et al.

1993, and references therein), the AraC family e.g.,

XylS (Inouye et al. 1986; Ramos et al. 1990), or the

NtrC family e.g., XylR and DmpR (Inouye et al. 1988;

Shingler et al. 1993). For a long time XylR was the

sole example of an NtrC-like regulator. However, as

described earlier, gene-probing experiments using a

dmpR

A-domain gene probe identified highly homolo-

gous DNA associated with the phenol catabolic genes

P. putida

U and five marine isolates (Nordlund et

al. 1993). Similarly, a regulatory region with high

homology to

xylR

has been located upstream of the

phenol catabolic genes of P.

putida

P35X (Ng et al.

1993). Therefore, the NtrC-like transcriptional activa-

tors promise to feature more prominently in the future

with respect to regulation of aromatic catabolism.

Concluding remarks

Current knowledge of

meta-cleavage

pathway enzy-

mology, and to some extent chemistry, lags behind

genetic characterization of the pathway. However,

analysis of the genes of the complete pathway, and

comparisons of the amino acid sequences of related

proteins, have identified potentially important residues

for many of these enzymes. Moreover, cloning and

over-expression of these genes can help provide large

quantities of the proteins for enzymological and struc-

tural studies. In the absence of structural data, the infor-

mation gained from genetic analysis provides a rational

basis to begin testing the importance of residues iden-

tiffed as, for example, potentially ligating prosthetic

groups, interacting with co-factors, or being involved

in catalysis. One caveat is that since a number of the

enzymes of the

meta-cleavage

pathway appear to be

unrelated to other proteins, and others are associated

with each other, it is particularly important to initially

characterize the enzymes from the parent strain before

proceeding to large-scale over-expression systems.

A fuller understanding of

meta-cleavage

pathway

enzymology also depends on better characterization of

the lower pathway chemistry. At the very least, bet-

ter methods for preparation of some of the enzyme

substrates are required (e.g., Powlowski et al. 1993).

In addition, since many of these substrates can exist

as keto-enol tautomers and/or

cis-trans

isomers, they

must be very carefully characterized. In this respect

the recent work of Whitman and colleagues with 4-

oxalocrotonate isomerase is exemplary (Whitman et

al. 199t; Chen et al. 1992).

One of the particularly interesting properties of

some of the

meta-cleavage

pathway enzymes, shown in

this and other work, is the formation of tight multien-

zyme complexes. In the

dmp

system tight associations

between the related polypeptides DmpH and DmpE

(4OD and OEH), and the unrelated DmpF and DmpG

polypeptides (ADA and HOA) have been identified

(see Fig. 2). In both cases the enzyme pairs catalyse

sequential steps of the pathway and the member of each

pair that makes the substrate for its partner is appar-

ently dependent on the presence of that partner for

activity (Shingler et al. 1992; Harayama et al. 1989).

In the case of the DmpF/DmpG pair, cross-regulation

of enzyme activity is possible (Powlowski et al. 1993).

These properties suggest that metabolic channelling

may be important for pathway efficiency, thus provid-

ing additional evolutionary impetus for clustering of

the genes involved into an operon. Co-purification of

enzyme activities through multiple steps of purifica-

tion only identifies tight associations and it may well

be that other enzymes of the pathway are also associat-

ed, albeit loosely. The existence of these associations,

and the so-far unproven idea of metabolite channelling,

certainly merit further attention.

The application of microbial metabolic activities to

detoxiffcation and clean-up has stimulated much inter-

est in construction of strains with improved degrada-

tive efficiency or expanded catabolic capacity. While

changes in substrate specificities of enzymes or effector

recognition by regulators have been successful, little

has been reported on genetic manipulation of strains

for improved efficiency. One possible strategy is simple

233

over-expression of the enzymes involved. The obser-

vation of comptexing between

meta-cteavage

pathway

enzymes has obvious practical implications for this

type of work. In addition, the cross-regulation evident

between the DmpF/DmpG pair highlights the fact that

very little is really known about (potential) regulation

at the enzyme level in this pathway. This will undoubt-

edly be remedied as progress on pathway enzymology

is made. Finally, the toxic effects of over-expression of

some of the enzymes (e.g., phenol hydroxylase) sug-

gest further hindrances to increasing strain efficiency

by brute force over-expression.

It is when manipulations for practical applications

are considered that the importance of a comprehen-

sive knowledge of pathway genetics, enzymology, and

chemistry is realized. The current boom in knowledge

of the genetics of the

meta-cleavage

pathways, and

their associated oxygenases, contributes enormously

to bringing this goal closer for at least one type of

model system.

Acknowledgements

We wish to thank our many colleagues, particularly P

Williams, K Kikuchi, PCK Lau, CL Poh, LC Ng, L

Sahlman, D Ahmad and M Gullberg for useful com-

ments, discussion and for providing information prior

to publication. Work in our laboratories was supported

by the National Swedish Research Councils for Nat-

ural Science and Engineering Science, The Natural

Science and Engineering Research Council of Canada,

and Concordia University.

Notes

1. In the original reference (Powlowski & Shingler

1990), the metal ion and NADH concentrations in

the

in vitro

assay was given in mlvI. This was a

typographical error, and the concentrations should

have read 100#M and 280#M respectively.

2. Computer searches of the EMBL/GenBank

(release 35) were made using the TFastA program

of the GCG Wisconsin sequence analysis software

package. One to one protein sequence alignments

were performed using the BestFit program.

234

References

Abril M-A, Michan C, Timmis KN & Ramos JL (1989) Regulator

and enzyme specificities of the TOL plasmid-encoded upper path-

way for degradation of aromatic hydrocarbons and expansion of

the substrate range of the pathway. J. Bacteriol. 171:6782-6790

Andrews SC, Shipley D, Keen JN, Findley JBC, Harrison PM

& Guest JR (1992) The haemoglobin-like protein (HMP) of

Escherichia coli

has ferrisiderophore reductase activity and its

C-terminal domain shares homology with ferredoxin NADP +

reductases. FEBS Lett. 302:247-252

Anonymous (1993) Facts and figures for the chemical industry.

Chem. Engineer. News 71:38-82

Assinder S & Williams PA (1990) The TOL plasmids: determinants

of the catabolism of toluene and xylenes. Adv. Microbial Physiol.

31:1-39

Ballou DP (1982) Flavoprotein monooxygenases. In: Massey V &

Williams CH (Eds) Flavins and Flavoproteins (pp 301-310). Else-

vier Science Publishing, Amsterdam

Bartilson M, Nordlund I & Shingler V (1990)Location and organi-

zation of the dimethylphenol catabolising genes of

Pseudomonas

CF600. Mol. Gen. Genet. 220:294-300

Bartilson M & Shingler V (1989) Nucleotide sequence and expres-

sion of the catechol 2,3-dioxygenase-encoding gene of phenol-

catabolizing

Pseudomonas

CF600. Gene 85:233-238

Bayly RC & Barbour MG (1984) The degradation of aromatic com-

pounds. In: Gibson DT (Ed) Microbial Degradation of Organic

Compounds (pp 253-294). Marcel Dekker, New York

Bayly RC & Wigmore GJ (1973) Metabolism of phenol and cresols

by mutants of

Pseudomonasputida.

J. Bacteriol. 113:1112-1126

Burton RM & Stadman ER (1953) The oxidation of acetaldehyde to

acetyl coenzyme A. J, Biol. Chem. 202:873-890

Carlson J, Fuchs JA & Messing J (1984) Primary structure of the

Escherichia coIi

ribonucleoside diphosphate reductase opemn.

Proc. Natl. Acad. Sci. U.S.A. 81:4294-4297

Carrington B, Lowe A & Williams PA (1994) The lower path-

way operon for benzoate catabolism in biphenyl-utilising

Pseu-

domonas

sp. strain IC and the nucleotide sequence of the

bphE

gene for catechol 2,3-dioxygenase. Microbiology 140:499-508

Chen LH, Kenyon GL, Curtin F, Harayama S, Bembenek ME,

Hajipour G & Whitman CP (1992) 4-Oxalocrotonate tau-

tomerase, an enzyme composed of 62 amino acid residues per

monomer. J. Biol. Chem. 267:17716-17721

Coco WM, Rothmel RK, Henikoff S & Chakrabarty AM (1993)

Nucleotide sequence and initial functional analysis of the

clcR

gene encoding a LysR Family activator of the

clcABD

chlorocat-

echol operon in

Pseudomonasputida.

J. Bacteriol. 175:417-427

Collinsworth WL, Chapman PJ & Dagley S (1972) Stereospecific

enzymes in the degradation of aromatic compounds by

Pseu-

domonas putida.

J. Bacteriol. 113:922-931

Dagley S (1975) A biochemical approach to some problems of envi-

ronmental pollution. Essays Bio.chem, 11:81-138

Dagley S (1986) Biochemistry of aromatic hydrocarbondegradation

in pseudomonads. In: Sokatch JR (Ed) The Bacteria, Vol 10 (pp

527-556). Academic Press, New York

Dagley S & Gibson DT (1965) The bacterial degradation of catechol.

Biochemistry 95:466-474

Davenport RC & Whitman CP (1993) Preliminary analysis of crys-

tals of 4-oxalocrotonate tautomerase, an enzyme composed of

unusually small monomers. J. Mol. Biol. 231:509-512

Derewenda ZS & Sharp AM (1993) News from the interface: the

molecular structure of triacylglyceride lipases. Trends Biochem.

Sci. 18:20-25

Entsch B, Ballou DP & Massey V (1976) Flavin-oxygen derivatives

involved in hydroxylation by p-hydroxybenzoate hydroxylase. J.

Biol. Chem. 251:2550-2563

Ericson A, Hedman B, Hodgson KO, Green J, Dalton H, Bentsen

JG, Beers RH & Lippard SJ (1988) Structural characterization

by EXAFS spectroscopy of the binuclear iron centre in protein

A of methane monooxygenase from

Methylococcus capsulatus

(Bath). J. Amer. Chem. Soc. 110:2330-2332

Feller G, Thiry M & Gerday C (1991) Nucleotide sequence of the

lipase gene

lip3

from the antarctic psychotroph

Moraxella

TA 144.

Biochim. Biophys. Acta 1088:323-324

Fennewald M, Prevatt W, Meyer R & Shapiro J (1978) Isolation of

IncP-2 DNA fromPseudomonasaeruginosa.Plasmid

1:t64-173

Fox BG, Surerus KK, Mt~nck E & Lipscomb JD (1988) Evidence for

a #-oxo-bridged binuclear iron cluster in the hydroxylase com-

ponent of methane monooxygenase. J. Biol. Chem. 263:10553-

10556

Froland WA, Andersson KK, Lee S-K & Lipscomb JD (1992)

Methane monooxygenase component B and reductase alter the

regioselectivity of the hydroxylase-catalyzed reaction. J, Biol.

Chem. 267:17588-17597

Green J & Dalton H (1985)Protein B of soluble methane monooxy-

genase from

Methylococcus capsulatus

(Bath). J. Biol. Chem.

260:15795-15801

Gurujeyalakshmi G & Oriel P (1989) Isolation of phenol-degrading

Bacillus stearothermophilus.

Appl. Environ. Microbiol. 55:500-

5O2

Happold FC & Key A (1932) The bacterial purification of gas-works

liquors. The action of liquors on the bacterial flora of sewage. J.

Hygiene 32:573-580

Harayama S, Kok M & Neidle EL (1992) Functional and evolution-

ary relationships amoung divers oxygenases. Annu. Rev. Micro-

biol. 46:565-601

Harayama S, Polissi A & Rekik M (1991) Divergent evolution of

chloroplast-type ferredoxins. FEBS Lett. 285:85-88

Harayama S & Rekik M (1993) Comparison of the nucleotide

sequence of the

recta-cleavage

pathway genes of TOL plas-

mid pWW0 from

Pseudomonasputida

with other

meta-cteavage

genes suggests that both single and multiple nucleotide substitu-

tions contribute to enzyme evolution. Mol. Gen. Genet. 239:81-

Harayama S, Rekik M, Ngai K-1 & Ornston LN (1989) Physical-

ly associated enzymes produce and metabolize 2-hydroxy-2,4-

dienoate, a chemically unstable intermediate formed in catechol

metabolism via

meta-cleavage

Pseudomonasputida.

J. Baete-

riol. 171:6251-6258

Hempel J & JBrnvall H (1989) Aldehyde dehydrogenases-structure.

In: Crow KE & Batt RD (Eds) Human Metabolism of Alcohol,

Vol II, Regulation, Enzymology and Metabolites of Ethanol (pp

77-88). CRC Press, Boca Raton, FL

Horn JA, Harayama S & Timmis KN (1991) DNA sequence deter-

mination of the TOL plasmid

xyIGFH

genes

Pseudomonasputi-

da:

implications for the evolution of aromatic catabolism. Mol.

Microbiol. 5:2459-2474

Howard PH, Boethling RS, Jarvis WE Meylan WM & Michalenko

EIvI (1991) Handbook of Environmental Degradation Rates.

Lewis Publishers Inc, Chelsea, MI

Inouye S, Nakazawa A & Nakazawa T (1986) Nncleotide sequence

of the regulatory gene

xylS

on the

Pseudomonas putida TOL

plasmid and identification of the protein product. Gene 44:235-

242

Inouye S, Nakazawa A & Nakazawa T (1988) Nucleotide sequence

of the regulatory gene

xyIR

of the TOL plasmid from

Pseu-

domonasputida.

Gene 66:301-306

Johansson J, Von Bahr-Lindstr6m J, Jeck R, Woenckhaus C &

J6mvall H (1988) Mitochondrial aldehyde dehydrogenase from

horse liver: correlation of the same species variants for both the

cytosolic and mitochondrial forms of an enzyme. Enr. J. Biochem.

172:527-533

Kedishvili NY, Popov KM, Rougraff PM, Zhao Y, Crab DW &

Harris RA (1992) CoA dependent methylmalonate-semialdehyde

dehydrogenase, a unique member of the aldehyde dehydrogenase

superfamily. J. Biol. Chem. 267:19724-19729

Kikuchi K, Yasukochi Y, Nagata Y, Koana T, Fukuda M, Yano K

& Takagi M (1993) Analysis of gene structure for biphenyl and

polychlorinated biphenys (PCBs) degradation in Pseudomonas

sp. KKS102 (Abstract). The Fourth International Symposium on

Pseudomonas: Biotechnology and Molecular Biology, Vancou-

ver, August 1993

Kimbara K, Hashimoto T, Fukuda M, Koana T, Takagi M, Oishi M

& Yano K (1989) Cloning and sequencing of two tandem genes

involved in degradation o f 2,3-dihydroxybiphenolto benzoic acid

in the polychlorinated biphenyl-degrading soil bacterium Pseu-

domonas sp. strain KKSI02. J. Bacteriol. 171:2740-2747

Kukor JJ & Olsen RH (1991) Genetic organization and regulation of a

meta cleavage pathway for catechols produced from catabolism o f

toluene, benzene, phenol, and cresols by Pseudomonas pickettii

PKO1. J. Bacteriol. 173:4587-4594

Kukor JJ & Olsen RH (1992) Complete nucleotide sequence of thuD,

the gene encoding phenol/cresol hydroxylase from Pseudomonas.

pickettii PKO1, and functional analysis of the encoded enzymes.

J. Bacteriol. 174:6518-6526

Kumagai H, Yamada H, Matsui H, Ohkishi H & Ogata H (1970)

Tyrosine phenol lyase I. Purification, crystalization, and proper-

ties. J. Biol. Chem. 245:1767-1777

Lau PCK, Wang Y, Rawlings M, Labb6 D, Bergeron H, Brousseau

R & Gibson DT (1993) Regulation of toluene degradation in

Pseudomonas putida F1 by an unusual sensor-regulator pro-

tein (Abstract) The Fourth International Symposium on Pseu-

domonas: Biotechnology and Molecular Biology, Vancouver,

August 1993

Lister J (1867) On a new method of treating compound fracture,

abscess, etc.: with observations on the conditions of suppuration.

Lancet (March 16):326-329

Loomes KM, Midwinter GG, Blackwell LF & Buckley PD

(1990) Evidence for reactivity of serine-74 with trans-(N, N-

dimethylamino)cinnamaldehyde during oxidation by the cyto-

plasmic aldehyde dehydrogenase from sheep liver, Biochemistry

29:2070-2075

Marcotte P & Walsh C (1978) Sequence of reactions which follows

enzymatic oxidation of allylglycine. Biochemistry 17:5620-5626

Mason JR & Cammack R (1992) The electron-transport proteins

of hydroxylating bacterial dioxygenases. Annu. Rev. Microbiol.

46:277-305

McGrew RE (1985) Encyclopedia of Medical History (pp 20-24).

MacMillan Reference Books, MacMillan Press, London

Menn F-M, Zylslm GJ & Gibson I3T (1991) Location and sequence

of the todF gene encoding 2-hydroxy-6-oxohepta-2,4-dienoate

hydrolase in Pseudomonasputida F1. Gene 104:91-94

Nakai C, Kagamiyama H & Nozaki M (1983) Complete nucleotide

sequence of the metapyrocatachase gene of the TOL plasmid of

Pseudomonas putida rot-2. J. Biol. Chem. 258:2923-2928

Neujahr HY &Gaal A (1973) Phenol hydroxylase from yeast, Purifi-

cation and properties of the enzyme from Trichosporon cutaneum.

Eur. J. Biochem. 35:386-400

Ng LC, Sze CC & Poh CL (1993) Cloning, nucleotide sequence

and expression of chromosomally-encoded phenol hydroxylase

degradative genes in Pseudomonasputida P35X (Abstract) The

235

Fourth International Symposium on Pseudomonas: Biotechnolo-

gy and Molecular Biology, Vancouver, August 1993

Nordlund I & Shingler V (1990) Nucleotide sequence of the meta-

cleavage pathway enzymes 2-hydroxymuconic semialdehyde

dehydrogenase and 2-hydroxymuconic semialdehyde hydrolase

from Pseudomonas CF600. Biochim. Biophys, Acta 1049:227-

230

Nordlund I, Powlowski J, Hagstrfm A & Shingler V (1993) Conser-

vation of regulatory and structural genes for a multicomponent

phenol hydroxylase within phenol-catabolizing bacteria that uti-

lize a meta-cleavage pathway. J, Gen. Mierobiol, 139:2695-2703

Nordlund I, Powtowski J & Shingler V (1990a) Complete nucleotide

sequence and polypeptide analysis of multicomponent phenol

hydroxylase from Pseudomonas sp. strain CF600. J. Bacteriol.

172:6826-6833

Nordlund E SjOberg B-M & Eklund H (1990b) Three-dimensional

structure of the free radical protein of ribonucleotide reductase.

Nature 345:593-598

Nordlund P, Dalton H & Eklund H (1992) The active site structure of

methane monooxygenase is closely related to the binuclear iron

centre of ribonucleotide reductase. FEBS Lett. 307:257-262

North AK, Klose KE, Stedman KM & Kustu S (1993) Prokayotic

enhancer-binding proteins reflect eukaryote-like modularity: the

puzzle of nitrogen regulatory protein C. J, Bacteriol. 175:4267-

4273

Nurk A, Kasak L & Kivisaar M (1991) Sequence of the gene (pheA)

encoding phenol monooxygenase from Pseudomonas EST100t:

expression in Escherichia coli and Pseudomonas putida. Gene

102:13-18

Nunn WP (1987) Two-carbon compounds and fatty acids as carbon

sources. In: Neidhardt FC, lngraham JL, Low KB, Maganasik

B, Schaechter M & Umbarger HE (Eds) Escherichia Coli and

Salmonella Typhimurium: Cellular and Molecular Biology (pp

285-301). American Society for Microbiology, Washington, D.C.

Omston LN & Yeh W-K (1982) Recurring themes and repeated

sequences in metabolic evolution. In: Chakrabarty AM (Ed)

Biodegradation and Detoxification of Environmental Pollutants

(pp 105-126). CRC Press, BocaRaton, FL

Pathak D & Oltis D (1990) The refined structure of dienelactone

hydrolase at 1.8 ,~. J. Mol. Biol. 214:497-525

Polissi A & Harayama S (1993) In vivo reactivation of catechol

2,3-dioxygenase mediated by a chloroplast-type ferredoxin: a

bacterial strategy to expand the substrate specificity of aromatic

degradativepathways. EMBO L 12:3339-3347

Powlowski J, Sahtman L & Shingler V (1993) Purification and

properties of the physically associated meta-cleavage pathway

enzymes 4-hydroxy-2-ketovalerate aldolase and aldehyde dehy-

drogenase (acylating) from Pseudomonas sp. strain CF600. J.

Bacteriol. 175:377-385

Powlowski J & Shingler V (1990) In vitro analysis of polypeptide

requirements of multicomponent phenol hydroxylase from Pseu-

domonas sp. strain CF600. J. Bacteriol. 172:6834--6840

Ramos JL, Stolz, A Reineke W & Timmis KN (1986) Altered effec-

for specificities in regulators of gene expression: TOL plasmid

x3'lS mutants and their use to engineer expansion of the range

of aromatics degraded by bacteria. Proc. Natk Acad. Sci. U.S.A.

83:8467-8471

Ramos JL, Michan C, Rojo E DwyerD & Timmis KN (1990)Signal-

regulator interactions. Genetic analysis of the effector binding

site of xylS, the benzoate-activated positive regulator of Pseu-

domonas TOL plasmid meta-cleavage pathway operon. J. Mol.

Biol. 211:373-382

236

Rheinwald JG, Chakrabarty AM & Gunsalus IC (1973) A trans-

missible plasmid controlling camphor oxidation in Pseudomonas

putida. Proc. Natl. Acad. Sci. U.S.A. 70:885-889

Rosenzweig AC, Fredrick CA, Lippard SJ & Nordlund P (t993)

Crystal structure of a bacterial non-haem iron hydroxylase that

catalyses the biological oxidation of methane. Nature 366:537-

543

Sala-Trepat JM, Murray K & Williams PA (1972) The metabolic

divergence in the meta cleavage of catechols by Pseudomonas

putida. Eur. J. Biochem. 28:347-356

Sala-Trepat JM & Evans WC (1971) The meta-cleavage of cate-

chol by Azotobacter species: 4-oxalocrotonate pathway. Eur. J.

Bioehem. 20:400-413

Shingler V, Bartilson M & Moore T (1993) Cloning and nucleotide

sequence of the gene encoding the positive regulator (DmpR) of

the phenol catabolic pathway encoded by pVI150 and identifica-

tion of DmpR as a member of the NtrC family of transcriptional

activators. J. Bacteriol. 175:1596-1604

Shingler V, Franklin FCH, Tsuda M, Holroyd D & Bagdasarian M

(1989) Molecular analysis of a plasmid-encoded phenol hydrox-

ylase from Pseudomonas CF600. J. Gen. Microbiol, 135:1083-

1092

Shingler V & Moore T (1994) Sensing of aromatic compounds by

the transcriptional activator

ofphenol-catabolisingPseudomonas

sp. strain CF600. J. Bacteriol. 176:1555-1560

Shingler V, Powlowski J & Marklund U (1992) Nucleotide

sequence and functional analysis of the complete phenoI/3,4-

dimethylphenol catabolic pathway of Pseudomonas sp. strain

CF600. J. Bacteriol. 174:711-724

Spain JC & Gibson DT (1988) Oxidation of substituted phenols by

Pseudomonasputida F1 and Pseudomonas sp. strain JS6. Appl.

Environ. Microbiol. 54:1399-1404

Spoelstra SF (1977) Degradation of tyrosine in anaerobically stored

piggery wastes and pig faeces, Appl. Environ. Microbiol. 36:631-

638

Stainthorpe AC, Lees V, Salmond GPC, Dalton H & Murrell JC

(1990) The methane monooxygenase gene cluster of Methyto-

coccus capsulatus (Bath). Gene 91:27-34

Stainthorpe AC, Murrell JC, Salmond GPC, Dalton H & Lees

V (1989) Molecular analysis of methane monooxygenase from

Methylococcus capsulatus (Bath). Arch. MicrobioL 152:154-159

Steele MI, Lorenz D, Hatter K, Park A & Sokatch (1992) Character-

ization of the mmsAB operon of Pseudomonas aeruginosa PAO

encoding methylmalonate-semialdehyde dehydrogenase and 3-

hydroxyisobutyrate dehydrogenase. J. Biol. Chem. 267:13585-

13592

Walsh C (1979) Enzymatic Reaction Mechanisms (pp 745-759).

Freeman and Company, San Francisco

Wasserfallen A, Rekik M & Harayama S (1991) A Pseu-

domonas strain able to degrade m-toluate in the presence of 3-

chlorocatechol. Bio/Technol. 9:296-298

Whitman CP, Aird BA, Gillespie WR & Stolowich NJ ( 1991 ) Chem-

ical and enzymatic ketonization of 2-hydroxymuconate, a conju-

gated enol. J. Amer. Chem. Soc. 113:3154-3162

Wierenga RK, Terpstra P & Hol WGJ (1986) Prediction of the occur-

rence of the ADP-binding/3-c~-fl fold in proteins using an amino

acid sequence finger print. J. Mot. Biol. I87:101-107

Wigmore G J, Bayly RC & Berardino D (1974) Pseudomonasputida

mutants defective in metabolism of the products of meta-fisson

of cateehol and its methyl analogues. J. Bacteriol. 120:31-37

Williams PA, Assinder SJ & Shaw LE (1990) Construction of hybrid

xylE genes between the two duplicate homologous genes from

TOL plasmid pWW53: comparison of the kinetic properties of

the gene product. J. Gen. Mierobiol. 136:1583-1589

Williams PA & Sayers JR (1994) The evolution of pathways for

aromatic hydrocarbon oxidation in Pseudomonas. This Volume

Yamamoto S & tshimura Y (1991) Dioxygenases and monooxyge-

nases. In: Kuby J (Ed) A Study of Enzymes (pp 315-344). CRC

Press, Boca Raton, FL

Yen K-M & Karl MR (1992) Identification of a new gene, tmoF, in

the

Pseudomonas mendocina KR 1 gene cluster encoding toluene-

4-monooxygena,se. J. Bacteriol. 174:7253-7261

Yen K-M, Karl MR, Blatt LM, Simon MJ, Winter RB, Fansset PR,

Lu HS, Harcourt AA & Chen KK (1991) Cloning and characteri-

zation of a Pseudomonas mendocina KR 1 gene cluster encoding

toluene-4-monooxygenase. J. Bacteriol. 173:5315-5327

You l-S, Ghosal D & Gunsahs IC (I 991) Nucleotide sequence analy-

sis of the Pseudomonasputida PpG7 salicylate hydroxylase gene

(nahG) and its 3t-flanking region. Biochemistry 30:1635-I641

Zylstra GJ & Gibson DT (1989) Toluene degradation by Pseu-

domonas Ft: nueleotide sequence of the todC1C2BADE genes

and their expression in Escherichia coli. J. Biol. Chem.

264:14940-14946

Widespread and active piezotolerant microorganisms mediate phenolic compound degradation under high hydrostatic pressure in hadal trenches

Article

Full-text available

May 2024

Phenolic compounds, as well as other aromatic compounds, have been reported to be abundant in hadal trenches. Although high-throughput sequencing studies have hinted at the potential of hadal microbes to degrade these compounds, direct microbiological, genetic and biochemical evidence under in situ pressures remain absent. Here, a microbial consortium and a pure culture of Pseudomonas, newly isolated from Mariana Trench sediments, efficiently degraded phenol under pressures up to 70 and 60 MPa, respectively, with concomitant increase in biomass. By analyzing a high-pressure (70 MPa) culture metatranscriptome, not only was the entire range of metabolic processes under high pressure generated, but also genes encoding complete phenol degradation via ortho- and meta-cleavage pathways were revealed. The isolate of Pseudomonas also contained genes encoding the complete degradation pathway. Six transcribed genes (dmpKLMNOPsed) were functionally identified to encode a multicomponent hydroxylase catalyzing the hydroxylation of phenol and its methylated derivatives by heterogeneous expression. In addition, key catabolic genes identified in the metatranscriptome of the high-pressure cultures and genomes of bacterial isolates were found to be all widely distributed in 22 published hadal microbial metagenomes. At microbiological, genetic, bioinformatics, and biochemical levels, this study found that microorganisms widely found in hadal trenches were able to effectively drive phenolic compound degradation under high hydrostatic pressures. This information will bridge a knowledge gap concerning the microbial aromatics degradation within hadal trenches. Supplementary Information The online version contains supplementary material available at 10.1007/s42995-024-00224-2.

Taxonomic identification, phenol biodegradation and soil remediation of the strain Rhodococcus sacchari sp. nov. Z13T

Article

Full-text available

Jun 2024
ARCH MICROBIOL

Phenols are highly toxic chemicals that are extensively used in industry and produce large amounts of emissions. Notably, phenols released into the soil are highly persistent, causing long-term harm to human health and the environment. In this study, a gram-positive, aerobic, and rod-shaped bacterial strain, Z13T, with efficient phenol degradation ability, was isolated from the soil of sugarcane fields. Based on the physiological properties and genomic features, strain Z13T is considered as a novel species of the genus Rhodococcus, for which the name Rhodococcus sacchari sp. nov. is proposed. The type strain is Z13T (= CCTCC AB 2022327T = JCM 35797T). This strain can use phenol as its sole carbon source. Z13T was able to completely degrade 1200 mg/L phenol within 20 h; the maximum specific growth rate was μmax = 0.93174 h⁻¹, and the maximum specific degradation rate was qmax = 0.47405 h⁻¹. Based on whole-genome sequencing and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, strain Z13T contains a series of phenol degradation genes, including dmpP, CatA, dmpB, pcaG, and pcaH, and can metabolize aromatic compounds. Moreover, the potential of strain Z13T for soil remediation was investigated by introducing Z13T into simulated phenol-contaminated soil, and the soil microbial diversity was analyzed. The results showed that 100% of the phenol in the soil was removed within 7.5 d. Furthermore, microbial diversity analysis revealed an increase in the relative species richness of Oceanobacillus, Chungangia, and Bacillus. Graphical abstract

Recycled by leaf inhabitants: Terrestrial bacteria drive the mineralization of organic matter in lake water

Article

Full-text available

May 2024

Terrestrial organic matter subsidizes aquatic food webs and plays an important role in carbon cycling in lake ecosystems, where it is decomposed mainly by microbes. However, the contribution of terrestrial and aquatic microbiomes on terrestrial carbon cycling and their effects on the biochemical fate of carbon has remained understudied. Therefore, we explored the microbial carbon utilization of three chemically differing leaf species in lake water in microcosms and quantified the biochemical endpoints of leaf carbon in CO2, CH4, and microbial biomass. Additionally, we identified microbial taxa responsible for leaf carbon recycling and studied the role of epiphytic and endophytic leaf microbiomes in microbial community succession in lake water. Microbially utilized leaf carbon was mainly respired (82.7 ± 1.4%), whereas a small proportion (17.1 ± 1.4%) was assimilated into biomass. Carbon from nitrogen‐rich alder leaves was taken up at the fastest rate, whereas birch leaf addition produced the highest concentrations of CH4, suggesting that leaf chemistry affects the decomposition rate and biochemical fate of carbon. In particular, terrestrial bacteria shaped the succession of aquatic bacterial communities. The addition of leaves resulted in the equal contribution of epiphytic and endophytic bacteria in the lake water, whereas epiphytic fungi dominated the fungal community structure. Our results suggest that terrestrial bacteria originating from terrestrial leaves influence the microbiome succession in lake ecosystems and play a key role in linking terrestrial carbon to an aquatic food web and determining the quality of carbon emissions that are released into the atmosphere.

AraC transcriptional regulator, aspartate semialdehyde dehydrogenase and acyltransferase: Three putative genes in phenol catabolic pathway of Acinetobacter sp. Strain DF4

Article

Full-text available

Mar 2024

Desouky Abdelhaleem

The objective of this study was to identify genes associated with the biodegradation of phenol by Acinetobacter sp. strain DF4 through the use of differential display (DD) methodology. The bacteria were grown in YEPG medium, and total RNA was extracted and analyzed using labeled primers to detect gene expression differences. Three distinctively expressed cDNA bands (ph1, ph2, and ph3) were identified, cloned, and sequenced. DNA analysis involved searching for open reading frames (ORFs), verifying results with the NCBI database, predicting promoter regions, and constructing phylogenetic trees using bioinformatics tools. The ph1 gene displayed a 97% identity with the AraC transcriptional regulator, suggesting its potential role in regulating the ortho-catabolic pathway of phenol. The ph2 gene showed a 98% identity with aspartate semialdehyde dehydrogenase, which is involved in phenol degradation. The ph3 gene had a 93% identity with acetyltransferase. Essential transcription factors, such as TATA, GTGTGT, CACA, and CTTTT, were detected, and the three genes promoter regions were predicted. This study successfully identified functional genes involved in the metabolism of cyclic chemicals, particularly phenol, using the DD technique. These findings provide insights into the biodegradation pathways of phenol by Acinetobacter sp. Strain DF4 and may contribute to the development of more efficient bioremediation strategies for phenol-contaminated environments.

Different Enzymatic Strategy to Degrade Carbamazepine by Rhodococcus zopfii and Leifsonia shinshuensis

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Feb 2024

Biovalorization of Olive Mill Wastewater Using Phenol Degrading Bacteria to Produce Biofertilizer

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Full-text available

Jan 2023

Biological treatments for olive mill waste cleanup using bacterial strains have proven to be effective and environmentally friendly. Three phenol-degrading bacterial strains identified as Lysinibacillus macroides, Lysinibacillus boronitolerans, and Brevundimonas olei were isolated from olive mill wastewater (OMWW) have high efficacy in degrading polyphenols. These strains exhibited both enzymes, catechol 1,2-dioxygenase and catechol 2,3-dioxygenase, responsible for degradation of phenolic compounds to varying degrees. Lysinibacillus macroides, rather than Lysinibacillus boronitolerans or Brevundimonas olei, have the highest catechol-dioxygenase enzyme activities. Various factors were optimized during the OMWW fermentation process to maximize the phenol degradation process. Maximum degradation rates were reached after 35 days at 20% dilution and pH ranged from 6 to 7 with starch and yeast reaching 1 g/l when added as carbon and nitrogen sources. In greenhouse experiments, the final fermentation product was evaluated as an organic biofertilizer. Bioassay data showed that the application of fermented OMWW significantly increases barley height, fresh and dry weight, nitrogen, potassium, and phosphorus contents compared to non-fermented barley, regardless of the type of bacteria used. The results of this study demonstrate that biologically fermented OMWW is proved to be economically beneficial to be used as an organic biofertilizer.

Biodegradation of carbamazepine by bacterial mixed culture: An enzymatic insight

Article

Full-text available

Jul 2022

A mixed culture of Leifsonia shinshuensis and Rhodococcus zopfii was assessed on its ability to degrade carbamazepine (CBZ). The mixed culture was found to metabolise CBZ by utilizing two enzymes, oxygen oxidoreductase and aldehyde oxidase. Correlation analysis showed that the degraded CBZ was strongly correlated (R 2 ≈0.9) with bacterial culture growth. However, only the concentrations of oxygen oxidoreductase is strongly correlated (R 2 ≈0.9) with both degradation of CBZ and bacterial growth, whereas the aldehyde oxidase concentration was less strongly correlated (R 2 ≈0.8) with both degradation of CBZ and bacterial growth. This suggests that the mixed culture adopted two different strategies simultaneously to metabolise CBZ to support growth and reduce toxicity.

DELINEATING PHENOL DEGRADING BACTERIA FROM COIR RETTING CONTAMINATED SOILS OF SOUTHERN REGIONS OF CHERTHALA

Article

Full-text available

May 2024

The coir retting industries in Southern regions of Cherthala provides a combative environment for the multiplication of specialized microbes with high phenol biodegradation potential. The present study aimed at isolation and characterization of phenol degrading bacteria from coir retting contaminated soils. After a series of screening enrichment techniques in MSPM at 37ºC, two bacteria were found to have the highest efficiency of pronounced growth in phenol stress conditions. Also, these bacteria have shown to be able to remove 22.5μM of phenol concentration that was measured by modified Mordaque et al, 1999 protocol at 24, 48 and 72 hours of incubation with a regular interval of 24 hours . The strong phenol degrading bacterial isolates were identified and the sequences were deposited in genbank as Lysini bacillus sp strain GAKVM (MT476862.1) and Paenibacillus sp strain. GA2KVM (MT509865.1). In the concentrations of 22.5μM of phenol, MT476862.1was able to degrade 74.44% and MT509865.1were able to degrade 74.77% within 72 hours of incubation at 37ºC.

Different Enzymatic Strategy to Degrade Carbamazepine by Rhodococcus zopfii and Leifsonia shinshuensis

Article

Jan 2024

This research reports on the efficiency of Rhodococcus zopfii and Leifsonia shinshuensis to degrade approximately 99.98 ± 0.20% carbamazepine (CBZ) within 5 days. The degradation of CBZ was strongly correlated (R2 ≥ 0.9) to bacterial growth in L. shinshuensis, but only moderate (R2 ≈ 0.6) in R. zopfii. Both oxygen oxidoreductase and aldehyde oxidase concentrations were positively and strongly correlated (R2 ≈ 0.9) to the degradation rate of CBZ in both cultures. However, when it comes to bacterial growth (CFU mL−1/h), only the culture of L. shinshuensis showed a strong positive correlation (R2 ≈ 0.9) to both enzyme concentrations, whereas the culture of R. zopfii only shows a moderate correlation (R2 ≈ 0.7). The degradation of CBZ by R. zopfii did not directly translate into higher bacterial growth. This demonstrated the possibility of different CBZ degradation strategies was being adopted by these two different bacteria. Hence, this piece of information is important to elucidate the mechanisms of carbamazepine degradation and sustainable treatment.

Alleviation of heat stress in electrostimulation assisted SBR treating thermophilic phenol wastewater: Performance, microbial community assembly, heat resistance mechanism and antibiotic resistance genes

Article

Mar 2024
PROCESS SAF ENVIRON

Protein B of soluble methane monooxygenase from Methylococcus capsulatus (Bath). A novel regulatory protein of enzyme activity.

Article

Full-text available

Dec 1985

An understanding of the mechanism of biological methane oxidation has been hampered by the lack of purified proteins. We describe here a purification protocol for the previously uncharacterized protein B of the soluble methane monooxygenase from the obligate methanotroph Methylococcus capsulatus (Bath). Soluble methane monooxygenase is a multicomponent enzyme consisting of a hydroxylase component, protein A, a reductase component, protein C, and protein B. All three proteins are required for monooxygenase activity. Protein B proves to be a low molecular weight (16,000) single subunit protein devoid of prosthetic groups. The protein is a powerful regulator of soluble methane monooxygenase activity, possessing the capacity to convert the enzyme from an oxidase to an oxygenase. Proteins A and C together catalyze the reduction of molecular oxygen to water, a reaction prevented by protein B. The uncoupling of soluble methane monooxygenase in this manner displays a number of novel features. First, the product of the uncoupled reaction is water, and second, the uncoupling is independent of substrate. Free hydrogen peroxide is not an intermediate in the reduction of oxygen by the incomplete methane monooxygenase enzyme complex. Finally, electron transfer can occur between protein C and protein A in the absence of protein B and protein B prevents the steady-state transfer of electrons in the absence of an oxidizable substrate, such as methane. It is demonstrated that oxygen reduction occurs at the active site of the hydroxylase component, protein A. A unifying mechanism, describing the interaction of the three proteins of soluble methane monooxygenase, is proposed.

Flavoprotein monooxygenases

Chapter

Full-text available

Dec 1984

David Ballou

Toluene Degradation by Pseudomonas putida F1

Article

Full-text available

Sep 1989

The nucleotide sequence of the todC1C2BADE genes which encode the first three enzymes in the catabolism of toluene by Pseudomonas putida F1 was determined. The genes encode the three components of the toluene dioxygenase enzyme system: reductaseTOL (todA), ferredoxinTOL (todB), and the two subunits of the terminal dioxygenase (todC1C2); (+)-cis-(1S, 2R)-dihydroxy-3-methylcyclohexa-3,5-diene dehydrogenase (todD); and 3-methylcatechol 2,3-dioxygenase (todE). Knowledge of the nucleotide sequence of the tod genes was used to construct clones of Escherichia coli JM109 that overproduce toluene dioxygenase (JM109(pDT-601]; toluene dioxygenase and (+)-cis-(1S, 2R)-dihydroxy-3-methylcyclohexa-3,5-diene dehydrogenase (JM109(pDTG602]; and toluene dioxygenase, (+)-cis-(1S, 2R)-dihydroxy-3-methylcyclohexa-3,5-diene dehydrogenase, and 3-methylcatechol 2,3-dioxygenase (JM109(pDTG603]. The overexpression of the tod-C1C2BADE gene products was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The three E. coli JM109 strains harboring the plasmids pDTG601, pDTG602, and pDTG603, after induction with isopropyl-β-D-thiogalactopyranoside, oxidized toluene to (+)-cis-(1S, 2R)-dihydroxy-3-methylcyclohexa-3,5-diene, 3-methylcatechol, and 2-hydroxy-6-oxo-2,4-heptadienoate, respectively. The tod-C1C2BAD genes show significant homology to the reported nucleotide sequence for benzene dioxygenase and cis-1,2-dihydroxycyclohexa-3,5-diene dehydrogenase from P. putida 136R-3 (Irie, S., Doi, S., Yorifuji, T., Takagi, M., and Yano, K. (1987) J. Bacteriol. 169, 5174–5179). In addition, significant homology was observed between the nucleotide sequences for the todDE genes and the sequences reported for cis-1,2-dihydroxy-6-phenylcyclohexa-3,5-diene dehydrogenase and 2,3-dihydroxybiphenyl-1,2-dioxygenase from Pseudomonas pseudoalcaligenes KF707 (Furukawa, K., Arimura, N., and Miyazaki, T. (1987) J. Bacteriol. 169, 427–429).

Recurring Themes and Repeated Sequences in Metabolic Evolution

Chapter

Jan 2018

Regulator and enzyme specificities of the TOL plasmid-encoded upper pathway for degradation of aromatic hydrocarbons and expansion of the substrate range of the pathway

Article

Dec 1989

The TOL plasmid upper pathway operon encodes enzymes involved in the catabolism of aromatic hydrocarbons such as toluene and xylenes. The regulator of the gene pathway, the XylR protein, exhibits a very broad effector specificity, being able to recognize as effectors not only pathway substrates but also a wide variety of mono- and disubstituted methyl-, ethyl-, and chlorotoluenes, benzyl alcohols, and p-chlorobenzaldehyde. Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase, two upper pathway enzymes, exhibit very broad substrate specificities and transform unsubstituted substrates and m- and p-methyl-, m- and p-ethyl-, and m- and p-chloro-substituted benzyl alcohols and benzaldehydes, respectively, at a high rate. In contrast, toluene oxidase only oxidizes toluene, m- and p-xylene, m-ethyltoluene, and 1,2,4-trimethylbenzene [corrected], also at a high rate. A biological test showed that toluene oxidase attacks m- and p-chlorotoluene, albeit at a low rate. No evidence for the transformation of p-ethyltoluene by toluene oxidase has been found. Hence, toluene oxidase acts as the bottleneck step for the catabolism of p-ethyl- and m- and p-chlorotoluene through the TOL upper pathway. A mutant toluene oxidase able to transform p-ethyltoluene was isolated, and a mutant strain capable of fully degrading p-ethyltoluene was constructed with a modified TOL plasmid meta-cleavage pathway able to mineralize p-ethylbenzoate. By transfer of a TOL plasmid into Pseudomonas sp. strain B13, a clone able to slowly degrade m-chlorotoluene was also obtained.

Biochemistry of Aromatic Hydrocarbon Degradation in Pseudomonads

Chapter

Dec 1986

Stanley Dagley

Facts and Figures for the Chemical Industry

Article

Jan 1995

Alcohol dehydrogenases — structure

Article

Jan 1989

Encyclopedia of Medical History

Article

May 1986
Hist Teach

The meta Cleavage of Catechol by Azotobacter Species

Article

Jun 1971
Eur J Biochem

Catechol was metabolized through 2‐hydroxymuconic semialdehyde by cell‐free extracts of benzoate‐grown Azotobacter Strains. Some properties of catechol 2,3 oxygenase preparations from Azotobacter vinelandii 206 are described. Two different enzymatic activities able to attack 2‐hydroxymuconic semialdehyde have been found in crude extracts from benzoate‐grown cells; one catalyses a hydrolytic release of formate from the semialdehyde and the other a dehydrogenation of this compound to 4‐oxalocrotonate. However, the low, non‐inducible levels of 2‐hydroxymuconic semialdehyde hydrolase activity appear negligible for metabolic purposes and the semialdehyde seems to be dissimilated almost exclusively via 4‐oxalocrotonate, by the action of a NAD ⁺ ‐dependent dehydrogenase, in Azotobacter strains. A tautomerase activity responsible for the interconversion of the enol and keto forms of 4‐oxalocrotonic acid was found in extracts from benzoate‐grown cells. 4‐Oxalocrotonate was stoicheiometrically converted to CO 2 and 4‐hydroxy‐2‐oxovalerate by a partially purified extract, with the transient formation of a compound that appears to be 2‐oxopent‐4‐enoic acid. The 4‐oxalocrotonate decarboxylase activity was stimulated by Mg ²⁺ or Mn ²⁺ ions and was inhibited by EDTA. Cell‐free extracts from Azotobacter strains converted synthetic 4‐hydroxy‐2‐oxovalerate to acetaldehyde and pyruvate. A reaction sequence, termed the 4‐oxalocrotonate pathway, for the dissimiation of catechol to acetaldehyde and pyruvate by Azotobacter species is presented. All the enzymes operative in this pathway were inducible, except the 4‐hydroxy‐2‐oxovalerate aldolase. The findings described here are discussed in connection with the two previously reported meta cleavage pathways for the oxidation of catechol in Pseudomonas strains.

Genetics and Biochemistry of Phenol Degradation by Pseudomonas sp. CF600

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