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THE Jounm~ cm BIOLOGICAL CHEMISTRY
Vol. 248, No. 5, Issue of Mmch 10, pp. 1725-1730, 1973
PTiTltea in
U.S.A.
Enzymatic Epoxidation
II. COMPARISON BETWEEN THE EPOXIDATION AND HYDROXYLATION REACTIONS CATALYZED
BY THE w-HTDROXYLATION SYSTEM OF
PXEUDOMON-4X OLEOVORANX
(Received for publication, October 18, 1972)
SHELDON W.
MAY AND
BERNARD
J.
ABBOTT
From the Corporate Research Laboratory, Esso Reseaxh and Engineeying Company, Linclen, New Jersey
07036
SUMMARY
The w-hydroxylation enzyme system of
Pseudomonas
oleouorans
has been shown by others to catalyze the methyl
group hydroxylation of alkanes and fatty acids. We have
recently reported that this same enzyme system also cata-
lyzes the epoxidation of alkenes. In the present study, a
comparison of the hydroxylation and epoxidation reactions
revealed that both require the three protein components
(reductase, rubredoxin, and hydroxylase) of the w-hydroxyl-
ation system. The substrate 1,7-octadiene is converted
to both 7,8-epoxy-1-octene and 1,2-7,8-diepoxyoctane,
whereas I-octene is oxidized to both 7-octene-l-01 and 1,2-
epoxyoctane. In addition, both 7,8-epoxy-1-octene and
1,2-epoxyoctane are oxidized by the enzyme system. Epox-
idation, as found previously for hydroxylation, requires
molecular oxygen, and NADPH does not substitute for
NADH in either reaction. Both reactions are inhibited by
cyanide and exhibit a similar pH dependence. A 1: 1 molar
stoichiometry was found between the amount of NADH
oxidized and the amount of 7,8-epoxy-1-octene produced
from 1,7-octadiene. In another experiment a 1: 1 stoichi-
ometry also was observed when the disappearance of the
substrate 7,8-epoxy-1-octene was correlated with the amount
of NADH oxidized. In the presence of excess 02, NADH,
reductase, and rubredoxin, a competition was observed be-
tween octane and 1,7-octadiene for the hydroxylase com-
ponent. Although it has not been possible so far to purify
the hydroxylase to homogeneity, this study indicates that
epoxidation and hydroxylation are mechanistically similar
and thus may involve the same species of activated oxygen.
An enzyme system consisting of three protein coml)oncnts has
been isolated from Pseudomonas
oleovorans
by Coon and co-
workers (l-6). In the presence of NADH and molecular oxygen,
this system catalyzes the w-hydroxylation of fatty acids and the
terminal hydroxylation of alkanes. The three protein compo-
nents have been identified as rubredoxin, reductase, and w-h)--
drosylase.
Rubredosin is an ironsulfur protein of molecular weight 19,000
containing I iron atom per molecule. This enzyme, which np-
pare&y funct’ions as au electron carrier in the system, has been
purified to homogeneity (5)) and its amino acid sequence has been
deterrnined (7). In the presence of reductasc and NADH, rubre-
doxin catalyzes the reduction of octyl hydroperoxide to l-oc-
tanol (8). However, hydroperoxides have not been identified as
free intermediates in the hydroxylation reaction.
The reductase component has also been purified to ho-
mogeneity. It is a flavoprotein of molecular weight 55,000 which
transfers electrons from NADH to rubredoxin.
In contrast, the
w-hydroxylase is relatively unstable and attempts to purify this
enzyme have so far been unsuccessful (4).
We recently reported that this same enzyme system catalyzes
the conversion of terminal olefins to their corresponding 1,2-0x-
ides (9). In view of the inhomogeneity of the hydroxylase com-
ponent, it secmcd possible that the hydroxylation and epoxidation
activities were completely unrelated. In order to clarify this
situation, a detailed investigation of the epoxidntion reaction was
undertaken, and the results were compared to those obtained
from studies of the hydroxylat,ion react.ion.
MATERIALS AND iUE’J?HODS
Octane, I-octcnc,
1,7-octadiene,
hcsane, hexadecanc, l-oc-
tanol, and 2-octanol n-ere purchased from various sources and
were of the highest grade commercially available. 1,2-Dihy-
droxyoctane was the generous gift of Dr. J. TV. Frankenfeld.
Inorganic salts were the commercially available rcagcnt grade
materials. ?&hloroperbcuzoic acid was purchased from Mathe-
son, and NADH (grade III) was from Sigma Chemical Company.
The components of the P. oleovorans w-hydroxylation system
were obtained from Drs. X. J. Coon and R. F. Royer and had
the following characteristics.
Rubredoxin was the (1 Fe) species
(mol w-t 19,500), purified by the method of Lode and Coon (5)
and stored frozen at a concentration of 3.7 mg per ml. Bacterial
reducmse was purified by the method of Ueda et al. (6) and
stored frozen in 10% glycerol at a concentration of 0.7 mg per ml.
Two different preparations of partially purified w-hydroxylase
were used. The first preparation was obtained after agarose
chromatography (4) and was stored in liquid nitrogen at a con-
centration of 0.6 mg per ml. The second preparation was ob-
taincd after the 25 to 30% (NH&S04 fractionation (4) and was
stored frozen at a couccntration of 50 mg per ml.
This material
was diluted with Tris buffer immediately before use.
Reaction Conditions-In a typical experiment, the reaction of
substrate with the w-hydrosylntion system was carried out as
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1726
follow. To I ml of 0.05 IRI Tris buffer (pI1 7.4) n-crc added 100
~1 (60 pg) of agarose-chro~uatogral,hcd l~ydrosylasc, 5 ~1 (3.5 ,~g)
of reductase, 50 ~1 (0.18 mg) of rubredoxin, and 0.2 pmoles of
SXDH. The reaction was then initiated by the addition of 0.6
pmoles of substrate in 10 ~1 of acetorrc, the latter serving to
solubilizc the hydrocarborr substrate (cj, Refcrrnce 4). Occa-
sionally, the concentrations of reactants were varied sonicwhat
in order to adjust appropriately the reaction rate (see “Results”).
The reactions wcrc carried out at 25”, and tlie oxidation of NAkDH
was follow-cd spectrophotometrically at 340 nnr. When gas
chromntographic analysis was desired, the total reaction t,inre was
varied according to the activity of the particular Irydrorylase
preparation, but was usually between 10 and 30 min. In the
stoichiometry experiments, where short reaction times arc de-
sirablc, the reactions mere terminated after 3 or 4 min (see “Re-
sults”). The omission of substrate in control esperimcnts rc-
sulted in a decrease in the rate of NADII oxidation to 20 to 50%
of that observed in the presence of substrate. The rat’e of this
“endogenous,” or uncoupled, oxidation \:aricd somewhat with
the particular hydroxylase preparation (cf. Reference 4).
Product Eztraction-The oxidation products of the w-hydrox-
ylation system were extracted from 1.0 ml of reaction mixture
wit,h 0.25 ml of hcxane. The hexane-water emulsion was broken
by centrifugation, and the upper hexane layer was retained for
gas chromatographic analysis. Diethyl ether, instead of herane,
was used for the extraction of 1,2-7,8-dieposyoctane.
For quantitative estimation of product concentration, 2-0~
tanol, at a final concentration of 0.21 mar, was added to the
hesane as an internal standard prior to extraction. The required
standard curves were then prepared as follows.
Various amounts
of 7,8-epoxy-l-octcne or I.-octanol were added to 1.0 ml of an
inactive enzyme solution and then extracted with 0.25 ml of
hexane which contained 2-octanol. The recovered hcsane layer
was then analyzed by gas chromatography. The ratio of the
peak area of cposidc or I-octanol to that of the internal standard
(2.octanol) was plotted as the function of the amount of eposide
or I-octanol added to the enzyme solut,ion, to give the standard
curves. These plots revealed that a linear relationship cxistcd
between the peak area ratios and the concentration of epoxide or
I-octanol. A comparison of thcsc standard (urves n-ith those
obtained by adding epoxide or I-octanol directly to hesane re-
vealed that essentially all of t,he epoxide or
I-octmol was IWOV-
ered by our extraction procedure. The standard curve rclxting
the concentrat,ion of 7,8-epoxy-l-octene to the ratio of peak areas
is illustrated in Fig. 1.
Gus Chromatography--The reaction products were scparat,ctl
by flarne ionization gas chromatography by using a stainless steel
column (20 ft
X
J,Q inch) packed with lOc/c Carbowas 20 ill on
SO/l00 Chromosorb W (.ipplied Sciences Laboratories, State
College, Pa.). A chromatogram which dcmonstmtos the scpara-
tion of several reaction products is sholvn in Fig. 2. The column
temperature was maintained isothermally at InO“, arid the in
jector and detector temperatures were 250”. The carrier gas
flow rate was 35 ml of heliuni per min, and the injection \-olume
n-as 0.5 ~1.
Product ~denti~cation--Euz?-lllatically produced products were
identified by retention time comparisons with authentic stall-
dards. This identification was supplcmcnt’ed by establishing the
prcsencc or absence of product peaks before and after bromination
or acid hydrolysis. Bromination was accoml~lishcd b\- adding
0.3 pl of bromine to 0.25 ml of hexane extract. The resultant
nddit,ion of bromine to double bonds removed unsaturated com-
pounds from the chromatograms. &oxide product,s rserc hy-
PI 7,8-EPOXY-I-OCTENE/lOO cc HEXANE
FIG.
1.
Standard curve relating the concentration of 7,s.epoxy-
1-octene to the ratio of peak areas measured for 7,8-epoxy-l-
octene and 2-octanol. Standard solutions were prepared by add-
ing various amounts of 7,8-epox:--1.octene to a solution of 3.3 ~1
of 2-octanol per 100 ml of hexnne. Details of the gas chrornato-
graphic analysis are described in the text.
0’
I I I I
I 1 I I
I I
.6 1.2 1.8 2.4 3.0 3.6 4.2 4.8 5.4 6.0
RETENTION TIME (minutes)
Fro. 2. Chromatogram demonstrating t,hc separation of l-
octanol? 2-octanol, 1,2-epoxyoctane, and 7,8-epoxy-1-octene.
ADDroxlmatelv 3.3 ~1 of each comuolmd were added to 100 ml of
h&ane, and ihe sdlution was analyzed by gas chromatography
as described in the text.
drolyzcd by- adding 5 ,~l of c~oncrntratrd II&O4 to 1.0 ml of tile
aqueous enzyme reaction mixture. Complete hydrolysis of
eposides owurred at roolm temperatnrc within 5 mill after the
addition of t,hc acid. Thr diol 1)roducts of epoxidc hydrolysis
were not detected, although gas chroillntogra~)l~)- columns which
were recommcndcd spccificall~~ for dial annlyscs were employed
(10).
Syniheses
1
,d-Epozyoctune-To
a solution
of 10 a (87 mmoles) of I-oc-
tene in 150 ml of ether lvcrc
adrlrd
30 g (174 mmolcs) of m-chloro-
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pcrbenzoic acid, and the mixture KIS stirred for 16 hours at room
t’empcrat,ure.
The reactioli mixture was washed four times with
ZOC,; K&O~, once with 10CC SazSO:, and twice with water, and
then dried over hlgS04. The ether was evaporated under a
strcxm of nitrogen, and the crude product was distilled to give
pure 1,2-epoxyoctanc, b.p. 55.5 to 56.5 at 9 mm (value in litcra-
ture, 61” at 15 mm (11)). The XXR spectrum was consistent
with the structure.
The product was stored desiccated at 4’.
7
,S-Epoxy-l
-octene-This compound was synthesized from 1,7-
octadiene using the same general procedure as described above,
except that a 1: 1 molar ratio of octadiene to m-chloroperbcnzoic
acid was used. The product n-as purified by distillation, b.p.
58.5 to 59.5 at 6.5 mm.
The SMR showed the expected ratio
of olcfin to epoxide protons, and 1~s consistent with the structure.
1,2-7 ,s-Diepoxyoctane-Tllis compound was synthesized from
1,7-octadienc and nz-cllloropelhenzoie acid as described above,
except that a 2.1: 1 molar ratio of perncid to diene was used, and
the reaction time was increased to 30 hours.
The product was
not distilled, but gas clllolllatogr:lphic analysis and NMR indi-
cated that the product was upprosimately 90 to 9576 pure.
RESULTS
Substrafe Reactivity-The
rates of NADH oxidation observed
wit’11 the w-hydrosylation system in the presence of several sub-
strates are presented in Table I. Table I shows that the
rclati1.e rates of NADI-I oxidation \Cth the various substrates
depended somewhat on the particular hydroxylase preparation
used, but in all cases 1,7-octndicnc, which has no methyl group
available for hydrosylntion, n-as t’he most reactive substrate.
Very little NADH oxitlatiou was observed with hesadecane, con-
firming
earlier reports that 1 his compound is a very poor substrate
for this enzyme system (4).
TABLE 1
Relative rates of NASH oxidnfion observed upon the addition of
various substrates to the w-hyclroxylntion enzyme system
The reactions of variolls substrates with the w-hydroxylation
system were carried out at
25”
as follows.
To 1 ml of 0.05
M
Tris
buffer (pH 7.4) were added 100 ~1 (GO pg) of agarose-purified hy-
droxylase, 5 ,LJ (3.5 gg) of reductase, 50 ,ul (0.18 mg) of rubredoxin,
and 0.2 ,umole of NADII.
The reaction was then initiated by the
addition of 0.6 Hmole of substrate in 10 ~1 of acetone.
Relative
rates are corrected for the endogenous oxidation of NADH.
Substrate
Octane..
LOctene
GS
79
1,7-Octadiene. _.
100 1
1,2-Epoxyoctane.
58
7,8-Epoxy-I-octene
63
Hcxadecane.
0 0
Relative rates of NADH oxidation (340 nm)
Trial0
Product detected by
* I u
~ C / D
gas chromntography
50
100
35
I-Octanol
1,2-Epoxyoctane
and 7-octene-l-01
7,8-Epoxy-I-octene
Not determined*
1,2-7,
%Diepoxy-
octane”
None
a Different trials rep’esent, results obtained on separate days
with hydroxylase preparations of various ages.
6 An authentic sample of the anticipated reaction product,
7,8-epoxy-l-octanol was not available for gas chromatographic
analysis.
I-Octanol
c To detect this product it n-as necessary to extract it from the
a These values are corrected for the endogenous oxidation of
reaction mixture with dict,hylether.
NADH.
The ohserred st’imulation of cofactor oxidation by l-octenc and
1 ,7-octadicne suggested that the w-hydrosylation system is capa-
ble of eposidating terminal double bonds. To confirm this possi-
bilit)-,
the
reaction 1)rotluct.s were extracted from the enzyme
solutions and annlyzcd by gas chromatography. The critcrin
for product identification \t-ere rctcntion time comparisons with
authentic standard:: and the presence or absence of the various
product, peaks after acid hydrolysis or bromination. ‘I’he brorni-
nation rcnction caust~l peaks representing compounds with double
bonds to disapljcar from the chromatogrnrns but
had
little effect
on saturated alcohols and cposidc pcnks. On the other hand,
acid 11).drolysi;; caused olrly those peaks rcpresentiug eposide
products to tlisalq)ear and had little effect on the other com-
pounds:. The retention times of the various products, and the
effects of the hytlrolysis and bromination reactions, were previ-
ously tabulated (9). From these analyses, it was found that
1 ,2-epoxyoctane was
lm~luccd
from I-octene, and 7,8-eposy-l-
octcne II-as produced from 1 ,7-octadiene (Table I). In addition
to 1,2-eposyoctanc,
7-octc11c-1-01
was forrned from I-octene by
the enzyme system. An authentic sample of 7-octene-l-01 was
not available for ret,c>lltion time comparisons. However, the
prcscnce
of this
compomrd was established on the basis of the
bromillntion and h;Vdl,olysis reactions, and the obscrvntion that
the retention lime of the new peak, as anticipated, was somewhat
greater than that of the peak representing I-octanol (SW Hefer-
ence 9). The diel)osidc product formed from 7 ,%cposy-l-octcne
could only he detectctl when the reaction misture was extracted
with ether inbtead of hesane.
Xfoichiol?letrll---li’or the dctcrmination of stoichiometry, the
amount of SADH oxidized was compared to the amount of l-oc-
tanol or 7,8-epoxy-l-octcne formed from octane and I ,7-octa-
diem, respectively. 12 1: 1 stoichiometry between cofactor
oxidation and product formation was obtained for both hydrox-
ylation of octane and cposidation of octadicne (Table II). In
these experiments, it was necessary to use short reaction times in
order to minimize the further oxidation of I-octanol and 7,X-
epoxy-l-octciir>. Whrn this precaution was not taken, a 1 : 1
stoichiomctry between cofactor oxidation and product formation
was not obtained for &her reaction.
A stoichiometry cq~criment was also performed by using 7,8-
epoxy-l-octene as the substrate,
since the diepoxide product
formed from thi.5 compound is not subject to further oxidation.
In this instance, cofactor oxidation was compared with substrate
loss, since the dic~poside product could not be quantitatively es-
Stoichiometry of NASH oxidation and product formation
for the w-hydroxylation system
The reaction conditions were as described in the legend to Table
I
except that 100 pg of (NK),S04-precipitated hydroxylase were
used, and, in the cpoxidation reaction, 1.5 pmoles of octadiene
Tyere added to initiate the reaction.
Cofactor oxidation was
measured spectrophotometrically for the indicated time period,
and the reaction mixtures lucre then assayed by quantitative gas
chromatography.
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172s
tracted using our standard procedure. After 1150 s of reaction
time, 130 nmolcs of NADH mere oxidized and 138 nmoles of 7, S-
epoxy-l-octenc reacted.
Component
Requirements-Tile various components of the w-
hydroxylation system were deleted individually in order to assess
the requirements for epoxidation of 1,7-octadiene. All compo-
nents of the system were necessary for maximal epoxidc produc-
tion (Table III). A small amount of eposide was produced when
the reductase was deleted; however, hydrosylation of octane also
proceeded to a comparable extent without the addition of re-
ductase.
As has been mentioned above, the w-hydrosylase used in this
study had not been purified to homogeneity. This fact most
likely accounts for our observation of a low level of both hydros-
ylation and epoxidation in the absence of added reductase since
a small amount of this component could be expected to bc present
in the w-hydroxylase preparat’ion, and very little rcductase is re-
quired for maximal a&iv&y.
Also, it has recently been reported
that purified rubredoxin occasionally contains a trace of re-
ductase (6).
Cofactor Specificity and Cyanide Inhibition-The data in Table
IV show the effect on both hydroxylation and epoxidation, of
substituting NADPH for NADH and of adding cyanide. Coon
and co-workers have previously reported that hydroxylation is
inhibited by cyanide (4) and is supported very ineffectively by
NADPH (2). It is apparent from our data that these same con-
clusions hold true for the epoxidative activity of the P. oleovorans
enzyme system.
Oxygen Requirement-The epoxidative activity of the w-hy-
droxylation enzyme system is greatly reduced under anaerobic
conditions, as shown in Table V. The fact that both NADH
and 02 are required for epoxidation strongly suggests t.hat the P.
oleovorans enzyme system is acting as a mixed function oxidase
in catalyzing this reaction.
pH Dependence-The
effect of pH on octadiene eposidation is
illustrated in Fig. 3. In these experiments, product formation
rather than cofactor oxidation was measured in order to eliminate
TABLE III
Effect of deleting components of the
w-hydroxylation enzyme system
on the production of l-octanol and Y,d-epoxy-1-octene
The reactions of octadiene or octane with the w-hydroxylation
system were carried out as described in the legend to Table I,
except
that various components were deleted as indicated below.
Initial rates of NADH oxidation were determined spectropho-
tometrically, and amounts of products formed were determined
by quantitative gas chromatography, as described under “Ma-
terials and Methods.”
System
Initial rate of
NADH oxidation
(with octadiene)a
Complete.
No substrate..
No hydroxylase.
No reductase..
No rubredoxin. .
No NADH. .
0.058
0.013
0.019
0.012
0.00
Relative
amounts of
7, “;;~w;,-l-
detected from
octadiene
oxidation
Relative
amounts of
I-octanol
detected from
octane oxidation
100 100
0 ND”
0
NDb
20
20
0
ND6
0
NDb
-
0 Absorbance change at 340 nm in 100 s.
b ND, not determined; see data in Reference 4 that establishes
the requirement for these components for hydroxylation.
complications which might arise from a 1~1-1 tlcpondence of en-
dogenous cofactor oxidation. The result:: are similar to those
reported earlier by McKenna and Coon (4) for the hydroxylat,ion
of octane. Both epoxidatipn and hydras>-lntion exhibit maximal
activity in Tris buffer near pH 7.5, but a shift of maximal activity
toward lower pH in phosphate buffer was more pronounced for
the hydrosylation reaction. However, it should be noted that
the data of McKenna and Coon were obtained by measuring co-
factor oxidation and not product. formation.
Substrate Competition-The data in Table VI show that the
total amount of product (l-octanol plus 7, S-epoxy-l-octme)
formed in the prcscncc of both octane and octadicne is less than
the
sum
of products formed from octane and octadiene alone.
Under the conditions of these experiments, h;r-droxylasc was the
limiting component and the concentrations of rubredoxin, re-
ductase, 02, and NADH were high enough to support the simul-
taneous oxidation of both octane and octadienr at the same rates
at which they occur indcpcndentlg. Therefore, the fact that
inhibition is observed suggests that hydrosylation and epoxida-
TABLL IV
Cofactor selectivity and cyanide inhibition jar h?ydroxylation
and epoxidation reactions
The reactions were carried out at 25” as follows. To 1 ml of
0.05 M Tris buffer (pH 7.5) were added 100 pg of (NH4)$Oa--pre-
cipitated hydroxylase, 3.5 pg of reductase, 0.18 mg of rubredoxin,
and 0.2pmole of pyridine nucleotidc. The reaction was initiated
by t.he addition of 0.6 rmole of substrate in 10 ~1 of acetone.
When included, NaCN was added immediately before the NADH.
SADH oxidation”
system
Substrate = Substrate =
octane octadiene
Complete + NADH. 16 20
Complete + NADPH..
1.2 i
2.3
Complete + 5 X 10m3 M
NaCN
+
NADH. , . . . ~. . . . . . . . . . . . . . . . . ~ 0
/
0.5
a Initial rate of NADH oxidation, measured spectrophoto-
metrically and corrected for endoqenous oxidation.
TABLE V
Oxygen requirement for epoxidation
The reactions were carried out as follows.
To 2 ml of 0.05 ~1
Tris buffer (pH 7.5) in a Thunberg cuvette, were added I mg of
(NH&SOd-precipitated hydroxylase, 7.0 pg of reductase, 0.35 mg
rubredoxin, and 1 pmole of NADH.
The cuvette was placed in
an ice bath, the top was sealed, and dry nitrogen was slowly bub-
bled through the solution for 45 min. A solution of octadiene in
acetone (0.06 M) was simultaneously purged with nitrogen while
immersed in a Dry Ice-acetone bath. The reaction was initiated
by the addition of 1.8 Fmoles of this octadiene in 30 ~1 of acetone
to the enzyme solution by means of a syringe. In the aerobic
control experiment, air was introduced after the addition of sub-
strate. After 7 min, the reaction mixture was analyzed by gas
chromatography.
Condition
nMoles of 7,8-epoxy-1-octene
formed per ml of reaction mixture
Aerobic ...........................
Anaerobic .........................
50
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90
1
80
70
8
I
;60
l-
2
t
w 50
>
i=
240
&
30
I
0'
r
I
I I I I
5.0 6.0 7.0 8.0 9.0 10.0
PH
FIG.
3. The effect of PI-I on the enoxidative activity of the
w-hydroxylation system.- The reactions were carried out as fol-
lows. To 1 ml of the appropriate buffer were added 0.2 mg of
(NH4)#04-precipitated hydroxylase, 90 pg of ritbredoxin, 3.5 pg
of reductase, 0.25 pmole of NADH, and 0.6 pmole of octadiene.
The reactions were stopped after 10 min and analyzed by quan-
titative gas chromatography as described in the text. Tris-ace-
tate (0.05 al) (A), Tris-chloride (A), and phosphate (0) buffers
were used as indicated. An activity of 100% corresponds to 60
nmoles of 7,8-epoxy-l-octene per ml.
TIBLE VI
Competilion of octane atad octadiene for the w-hydroxylase
The reactions were carried out as follows. To 1 ml of 0.05
M
Tris buffer (pH 7.5) n-ere added 80 pg of (NH1)2S04-precipitated
hydroxylase, 7 fig of reductase, 0.14 mg of rubredoxin, and 0.2
@mole
of NAD1-T. The reaction was initiated by the addition of
1.5 Mmoles of substrate in 50 ~1 of acetone. In the combination
reaction, 1.5 rmoles of each substrate was added in a total of 50
~1 of acetone. The reactions were stopped after 10 min and ana-
lyzed by quantitative gas chromatography.
Sutlstrate
Amount of product detected
1.Octanol 1 i,S-Epoxy-1-octene
nntoles
Octane ........................
7.5
Octadiene. ....................
11.0
Octane + oct,adiene.
.......... 0.8
8.5
tion are not associated with two independent enzyrncs present in
the hydrosylase preparationsi
1 An alternative explanation of this result is the following.
Distinct hvdroxylase and epoxidase enzymes do, in fact, exist
in our preparations. However, the enzyme which hydroxylates
octane is strondv inhibited bv octadiene, and, similarly, the en-
-1
zyme which epoxidates octadikne is strongly inhibited by octane.
In view of the apparent mechanistic similarity between hydroxyla-
tion and epoxidation, we feel that it is unlikely than an enzyme
capable of binding both octane and octadiene strongly will react
selectively with only one of these substrates.
1729
DISCUSSIOS
The results of the espcrirncnta described above establish
that
the w-hydrosylation system of P. oleovorans is capable of catalyz-
ing the cposidation of olefins as well as the hydrosylation of
alkanes. The rates
of
el)osidation and hydrosylation arc similar,
and all of the components of the system, plus NADH and molecu-
lar oxygen, are required for either reaction to occur. In addition,
both reactiotis UC inhibited by cyanide, affected similarly by pH,
and show the same cofactor selcctivitics. Tliesc facts suggest
that eposidation and hydrosylation proceed via similar mecha-
nisms.
Since the w-hydrosylase used in these stud& was not homo-
geneous, it is conceivable that hydroxylation and eposidation,
although mechanistically similar, arc actually associated with two
distinct enzymes present in our hydrosylasc preparation. This
possibility can be unequivocally eliminated only by complete
purification of the hpdrosylasc; all attempts to do so have so far
been unsuccessful. Kcverthclcss, the results of this study show
that, if such distinct “hydroxylasc” and “cposidase” enzymes
exist, they must be very similar.
The rclativcly high reactivity of octadienc (Table I) in this
system indicates that the absence of a terminal methyl group on
the hydrocarbon substrate does not prevent its binding to the
enzyme.
Thus, if t!m same w-hydrosylasc is, in fact, involved in
both eposidation and hydrosylation, the critical specificity ve-
quiremcnt for oxidizable hydrocarbon substrates may well b?
the presence of a mcthylenc chain. This, in turn, may account
for the reported resistance
of
highly branched hydrocarbons to
microbial degradation (12-14)) since the initial hydroxylation
rcactiou would not bc espectcd to occur readily with such sub-
strates.
The metabolic significance of eposidation by the
P. oleovorans
w-hydrosylation system is presently unclear. Abbott and Hou
have found in whole cell studies with another strain of
Pseudo-
monas
that both 1-octcne and 1,7-octadiene are eposidated, but
only I-octcnc will support grrowt.ll.2 The inability of 1 ,7-o&-
dicne to support growth indicates that this organism cannot
degrade the eposide functionality. If this is true for
P. oleo-
vorans
as well, terminal olefins such as I-octene, which are con-
verted to both hydrosylated and cposidated products, are metab-
olized esclusivcly via the hydroxylated end of the molecule.
C=C---(CH?)5-CH3 --f C ----C-(CH2)5--CH3
I
1
‘0’ i
C=C-(CHZ)~--CH~ ---$ C------ C-(CHa)r,-CHzOH
I
OH
‘0’
Thus, the role of the alkcnc eposidation reaction is not apparent.
There is currently considerable speculation as to the nature of
the activated osygen species which is involved in reactions
catalyzed by osygcnases. Strobe1 and Coon (15) have reported
that hydrosylations catalyzed by a rcconstitutcd liver microsomal
cytochrome I’-450 system arc supported by a superoside generat-
ing system and inhibited by supcrosidc dismutase, and similar
observations with intestinal tryptophan 2,3-dioxygenase have
been reported by Hirata and Hayaishi (I@. In contrast, no such
findings have been reported wit,11 the
P.
oleovorans system, and in
preliminary espcriments, not described here, we have been un-
successful in supporting either hytlrosylation or eposidation by
this enzyme system with a snuthine osidase-based superoxide
2 B. J. ilbbott and C. T. Iloll, iinpublished results.
by guest, on March 18, 2013www.jbc.orgDownloaded from
1730
generating system, or in inhibiting either reaction with supcroside
dismutasc. On the basis of these results and because of the
questionable metabolic significance of epoxidation, it is tempting
to speculate that the cpoxidation reaction may represent a trap
ping of the as-yet-undefined active oxygen spccics, ‘inornlally”
involved in hydrosylation, by the relatively rcactivc r-electron
system of the olefinic double bond. The notion that such a
species of active oxygen must then possess the correct electronic
and symmetry characteristics for insertion in either C-I-1 or
C=C bonds3 may be helpful in defiiiing the nature of this active
form of oxygen.
.IcknowZedgment-We thank Drs. M. J. Coon and R. I?. Bayer
for supplying the enzymes used in this study and for many helpful
discussions. WC are also grateful to Messrs. -1. Felix and C. J.
l\#IcCoy for escellcnt technical assistance.
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