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INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 12: 415-422, 2003
Inhibition of lipoxygenase by (-)-epigallocatechin gallate:
X-ray analysis at 2.1 Å reveals degradation of EGCG and
shows soybean LOX-3 complex with EGC instead
EWA SKRZYPCZAK-JANKUN1, KANGJING ZHOU1,3 and JERZY JANKUN1,2
1Department of Urology, Urology Research Center, Medical College of Ohio, 2Department of Physiology
and Molecular Medicine, Toledo, OH 43614, USA
Received May 18, 2003; Accepted July 24, 2003
Abstract. Lipoxygenases (LOXs) are non-heme iron containing
enzymes ubiquitous in Nature and participating in the
metabolism of the polyunsaturated fatty acids (PUFA). They are
capable of combining their dioxygenase activity with its co-
oxidative activity manifesting itself in biotransformation
reactions catalyzed by LOXs for other than PUFA small
molecules. LOXs involvement in inflammatory diseases and
cancer have been well documented. Catechins are the natural
flavonoids of known inhibitory activity toward dioxygenases
with a potential to be utilized in diseases prevention and
treatment. This work presents results obtained from an X-ray
analysis of (-)-epigallocatechin gallate (EGCG) interacting with
soybean lipoxygenase-3. The 3D structure of the resulting
complex reveals the inhibitor depicting (-)-epigallocatechin that
lacks galloyl moiety. The A-ring is near the iron co-factor,
attached by the hydrogen bond to the C-terminus of the enzyme,
and the B-ring hydroxyl groups participate in the hydrogen
bonds and the van der Waals interactions formed by the
surrounding amino acids and water molecules.
Introduction
Dioxygenases (cyclooxygenases (COX-1, COX-2) and
lipoxygenases (5-LOX, 8-LOX, 12-LOX, and 15-LOX)) are
responsible for the metabolism of the fatty acids (FA) and their
metabolites cause inflammatory responses in the body. They also
play significant role in cancer cell growth, metastasis,
invasiveness, cell survival and induction of tumor necrosis factor
(TNF) (1, 2). It has been pointed out, that the dietary FA enhance
cytotoxicity of some neoplastic agents and the anticancer effects
of radiotherapy (3).
___________________________________________
Key words: catechins metabolism, lipoxygenase, co-oxidation,
chemoprevention, X-ray structure.
Present address: Institute of Biotechnology, Fozhou University,
Fozhou, Fujian 350002, China
Correspondence to: Dr. Ewa Skrzypczak-Jankun, Urology
Research Center, Medical College of Ohio,
Toledo OH 43614-5807, Phone 419-383-5414, fax 419-383-
3785, e-mail: eskrzypczak@mco.edu
Popular anti-inflammatory drugs are usually COX-specific and
ineffective toward LOXs, and although they block one pathway
of FA metabolism, the other remains open. While there are only
two cyclooxygenase isozymes, COX-1 and COX-2 (see also ref.
43) the variety of LOXs and their products indicate much greater
complexity. For instance: LOXs involvement in cancer has been
well documented and summons our attention to all types of
LOXs and many of their products. Inhibition of the 5-LOX
pathway has a chemopreventive effect in animal lung
carcinogenisis, and blocks the oxidation of several potent
carcinogens (4, 5). 5-HETE has the ability to stimulate growth of
lung cancer cells, and prostate cancer cells (6, 7). The inhibitors
of 5-LOX have the ability to decrease cell proliferation and
trigger apoptosis (8). 8-LOX product, 8(S)-HETE plays role in
keratinocyte differentiation 12(S)-HETE, the major metabolite of
the 12-LOX pathway, has been shown to correlate with
metastatic potential and stimulate the expression of integrin
receptors leading to increased tumor cell adhesion (9, 10). Also,
12(S)-HETE can activate protein kinase C (PKC), which
mediates the secretion of cathepsin B, a cysteine protease that
has been shown to be involved in tumor metastasis and invasion
of colon cancer cells (11). In prostate cancer patients, elevated
12-LOX mRNA levels were shown to correlate with poor
differentiation and cancer cell invasiveness (12). Additionally,
12-LOX in human prostate carcinoma stimulates angiogenesis
and tumor growth (13). Recently it was shown that 5-HETE and
12(S)-HETE directly stimulate pancreatic cell proliferation and
that LOX inhibitors can induce apoptosis and cell differentiation
(14). 15-LOX and its products have been linked to cell
maturation and differentiation (15-17). This short review clearly
shows that understanding the mechanism of inhibition of LOXs
can have profound effect in the development of many anti-cancer
and anti-inflammatory drugs. On the basis of the available LOX
data it was suggested, that a combination of LOX modulators
might be needed to shift the balance of LOX activities from
procarcinogenic to anticancerogenic as a novel strategy for
cancer chemoprevention (Shureiqi I. Symposium 26:
Cyclooxygenase, lipoxygenase: Targets for the prevention and
Treatment of Cancer. Talk: Lypoxygenase modulation and
NSAID chemoprevention. In: American Association for Cancer
Research 93rd Annual Meeting, San Francisco, CA, 2002, pp.
1178).
416 SKRZYPCZAK-JANKUN et al: EGCG AND LOX
Catechins are described as colorless, astringent, water-soluble
polyphenols found in many fruits and grains, such as coffee, red
grapes, prunes and raisins. Their main source however comes
from a beverage made from tea leaves of Camellia Sinensis. (-)-
Epigallocatechin gallate (EGCG) together with other galloylated
catechins constitute more than 90% of the total catechin content
in green tea (18, 19). Epidemiological studies into the protective
effect of tea against human cancers have been reported as
inconclusive (20), but – on the other hand - occurrence of cancer
is significantly lower in the societies where green tea is always
present in the daily diet (21, 22). Laboratory studies strongly
indicate that tea inhibits certain cancers, and there is a multitude
of evidence confirming the anticancerogenic properties of the
individual catechins. For instance: EGCG alone shows
anticancer effectiveness against carcinogen-induced skin, lung,
forestomach, esophagus, duodeum, liver and colon tumors in
rodents. It was found to cause apoptosis and/or cell cycle arrest
in human carcinoma cells of skin and prostate cancers (23).
While synthetic drugs are developed mostly as highly specific,
the natural compounds usually target more than one enzyme.
Polyphenols are listed among other phytochemicals as inhibitors
of dioxygeneses with catechin(s) showing activity against both
COX and LOX (24). These nutraceuticals have been considered
as potential drugs less toxic than NSAIDs (Mortality because of
the NSAIDs toxicity is as high as from AIDS, >16,000/year -
Hawk E.T., AACR 93rd Annual Meeting, Symposium 26:
Cyclooxygenase, Lipoxygenase: Targets for prevention and
treatment of cancer. San Francisco, 2002). There are indications
that inhibition of LOX and COX is more effective than
inhibition of either pathway alone (4) and so multi-targeting may
be a much better approach (25). Presence of catechins and their
metabolites have been well documented in the human body and
in the animal models (26) but we still lack knowledge about
molecular aspects of their utilization.
Designing agents to modulate activities of the variety of so
closely homologous enzymes like different LOXs require an
intimate knowledge about their 3D structures, as well as
information about metabolism of the potential xeno- or
endobiotics. So far only the structures of soybean isozymes
LOX-1 and LOX-3 have been determined for native enzymes,
and several structures of their and rabbit 15-LOX (from
reticulocytes) molecular complexes with inhibitors are known
(see Discussion). Our previous experiments with curcumin (27)
and quercetin (28) documented LOX catalyzed oxidative
degradation of these polyphenols. Here we report soybean LOX-
3 interaction with EGCG and the X-ray analysis of the resulting
complex that reveals inhibitor with a missing galloyl moiety.
Materials and methods
Materials: Protein has been isolated from the soybean seeds cv.
Resnick, purified and crystallized according to the protocol
described in (29). EGCG was a gift from Dr. L.R. Juneja, the
commercial name Sunphenon GE-95, Nutritional Foods
Division, Taiyo Kagaku Co., Ltd., Akahori-Shinmachi,
Yokkaichi, Mie, 510-0825, Japan. Other chemicals were
purchased from Sigma or Fisher Scientific (Springfield, NJ) and
were of the analytical grade quality.
X-ray analysis: EGCG inhibitor was added to the crystals storing
solution in 100:1 molar ratio relatively to protein (in ethanol to
increase solubility)
Table I. Data collection and refinement statistics
____________________________________________________
Unit cell: C2, a=112.8, b=137.3, c=61.9 Å, β=95.6°, Z=4
Resolution, last shell (Å) 2.10 (2.18-2.10)
Unique reflections 43096
Completeness overall, last shell (%) 93.8, 94.1
Rmerge overall, last shell (%) 0.08, 0.36
Refinement (X-plor)
Protein, inhibitor, water (except H) 6779, 22,512
R, Rfree (F >2 σ(F) ) 0.199, 0.295
rms deviation from ideal geometry
Bond lengths (Å) 0.008
Bond angles (º) 1.63
Dihedral angles (º) 24.80
Improper angles (º) 1.39
PDB deposit 1JNQ
____________________________________________________
and soaked in for 22-60 hours before collecting data. All data
were taken on Raxis IV with Cu rotating anode and focusing
mirrors, for 2º oscillations at 140mm distance and 10-12min of
exposure per frame. Crystals soaked in EGCG did not show any
change of color. Data collected from 3 crystals (from the same
batch) were scaled and merged together to achieve completeness
and desirable intensity statistic. Coordinates of the native
enzyme (PDB code 1LNH) were used as a model for the
molecular replacement and the calculated maps clearly revealed
the outline of the inhibitor and its chirality. Chain ver.7 and
InsightII ver.98 (30, 31) were used for model rebuilding and
examinations against maps, on SGI workstation Indygo2
Extreme running under Irix ver. 6.5. The models fitted to the
electron density maps were refined against the experimental data
using programs X-plor ver. 3.85 or CNS ver.1.0. (32, 33).
Results are summarized in Table I and presented in Fig. 2-5.
Results and Discussion
Due to lack of sufficiently purified human enzymes most of the
structural research has been done on soybean LOX. In our
studies we have used soybean isozyme LOX-3. Despite the
difference in the number of amino acids between plant and
mammalian LOXs (~800 vs ~600), these proteins are amazingly
similar in topology with high similarities in the active site of
these enzymes (Fig. 1a). Comparison of the 3D structures of
LOXs complexes described so far shows that despite their
different chemical nature the inhibitors position themselves in
the same location, i.e. in the central cavity of the enzyme, where
the sixth ligand would be in the pseudo-octahedral coordination
sphere of the iron co-factor. The figures in this publication were
made using the following data (PDB entries given as four letter
code): (i) ‘Purple’ soybean LOX-3 with ferric iron: 1IK3 - with
metabolite of the linoleic acid, 13(S)-hydroperoxy-9,11-
cis,trans-octadecadienoic acid (13HPOD), 1HU9 – with product
of curcumin degradation, 4-hydroperoxy-2-methoxyphenol
(4HM), and with cumene peroxide (Skrzypczak-Jankun E., et al.
"Purple" lipoxygenase - X-ray analysis of complexes with three
INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 12: 415-422, 2003 417
A
B
Fig. 1 A Superimposition of mammalian (rabbit) and plant (soybean) LOXs with different inhibitors. B, Superimposition of
soybean LOX-3 enzyme (‘purple’ from) with different peroxides.
different peroxides. In: American Crystallographic Association
Annual Meeting, St. Paul, July 22-27 2000, pp. 37-02.01.02.).
(ii) Soybean LOX-3 with ferrous iron: 1BYT – with 4-
nitrocatechol, 1N8Q – with protocatechuic acid, product of
quercetin degradation, and 4-thia-2-decenal (Skrzypczak-Jankun
E., et al. Soybean lipoxygenase complexes with small molecules.
In: American Crystallographic Association Annual Meeting,
Arlington, July 18-23 1998, pp. 99-P040). (iii) Rabbit 15-LOX
with aryl carboxylate inhibitor RS75091 – 1LOX. While
peroxides (Fig. 1b) are bound to the ferric ion the other inhibitors
418 SKRZYPCZAK-JANKUN et al: EGCG AND LOX
Quercetin Protocatechuic acid (DHB)
OH
OH
COOH
O
OH
OH
OH
OH
O
OH
O
OH
OH
OH
OH
O
OH
OH
OOH
OH
AC
B
D
O
OH
OH
OH
OH
OH
OH
1
2
3
4
5
7
3'
4'
6
8
5'
6'
2'
1'
9
10
EGCG EGC
OOH
OH
OCH3
OH
H3CO
OH
O
OOH
CH3
O
O
O
CH3
Curcumin 4HM
Fig. 2. X-ray analysis of soybean LOX-3 in complexes with its inhibitors: curcumin, quercetin and EGCG shows degradation of natural flavonoids.
appear in the X-ray analysis at non-bonding distance to the
ferrous ion. In case of our experiment with EGCG we have
found the inhibitor molecule at the same location but without
galloyl moiety (ring D –C(=O)- ) – Fig. 2, 4a, which corresponds
to EGC.
EGCG or EGC? Alhough the soaking experiment was done with
EGCG, the X-ray structure does not show galloyl moiety
suggesting hydrolysis of the ester. The electron density clearly
outlines the molecule and defines chirality for atoms C2 and C3.
Incorporating the whole EGCG molecule would require shift not
only in the side chains but also in the main chain positions of
residues Gln514, Leu515 and Leu565. Another possibility would
be placing the D ring near Trp519, or toward Phe576 (Fig. 3),
also upon some movement of the nearby residues for better
accommodation. Neither model could be convincingly confirmed
in the refinement and so the structure is reported true to the
electron density map as the complex with EGC, a result of the
co-oxidative activity of LOX (34). (-)-Epigallocatechin makes
the following interactions: O1 points toward Trp519, O3…H-
CG1 Ile572, 2.7 Å, O5…H-CD1 Ile773, 3.0 Å, O7…Fe, 2.7 Å,
and 2.5 Å to OT1 Ile 857(C-terminus), O3’ points toward
Phe576, O4’…Water2004…O Ser510, 2.9 Å, 2.6 Å respectively,
O5’…OD1 Gln716 ND2…OD1 Asn713, 2.4 Å, 2.5 Å
respectively.
Catechin degradation. The products of EGCG degradation have
been studied extensively. Zimeri & Tong published the results of
chemical studies about degradation kinetics of EGCG as a
function of pH and dissolved oxygen in a liquid model systems
(18). Their aqua’s media have different amounts of dissolved
oxygen, and were buffered by citrate buffer at pH 4,5,6,7. The
measurements determined the ratio of EGCG concentration at a
given time in relation to the starting value as a function of (i)
temperature at 30, 80 90 and 100ºC, (ii) pH, and (iii) amount of
the dissolved oxygen. The authors did not characterized the
products of degradation but all their data indicate rapid
decomposition of EGCG and only <10% of the compound left
beyond 10 hours at pH 7 and 30ºC. Ho and co-workers have
found different oxidation products depending on the reaction
conditions and concluded that different oxidants can produce
different products of catechin oxidation. The reaction with
horseradish peroxidase and H2O2 in aceton:water mixture(35)
produced a symmetric homo-dimmer of EGCG as its oxidation
product, where the B rings lose their aromatic character and get
triply connected in C4’-C6’, C5’-O-C5’ fashion, with C1’-C2’-
C3’-OH changing to C1’=C2’-C3’=O (Fig. 5 – 1). Similar
reaction conditions for mixture of EGC and EGCG but with less
aceton and mixed with pH 5 citrate buffer, brought another
molecule with benzotropolone fragments (Fig. 5 – 2 (36)). 2,2-
Diphenyl-1-picrylhydrazyl radical (DPPH) gives dimmer
connected by C2’-C2’ (Fig. 5 – 3 (36)). FeSO4 and H2O2 or H2O2
alone bring yet another different products, EGCG A-ring
cleavage metabolites – 4 and 5 (35). Kondo et al. studied the
effect of catechins on lipid peroxidation in soybean
phosphatidylcholine liposomes, initiated by 2,2’-azobis(2-
aminopropane)-hydrochloride.
INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 12: 415-422, 2003 419
Fig. 3. EGCG fitted into LOX-3. The doted ball marks the iron coordination sphere of 2 Å radius. Galloyl ring (D) from EGCG can be placed near
Trp519 or near Phe576. In either case it collides with the hydrophobic residues (selected residues colored gray) and there is no density in the electron
density maps beyond O3 to support the presence of this ring.
Fig. 4a. A fragment of the electron density map 2Fo-Fc contoured at 1σ, EGC represented in yellow, the sequential numbers given for the selected
residues. Others: above Fe and His518, to the left – Ile773, below EGC – Trp519, to the right and below Ile557 - Asn558.
Fig. 4b. Same fragment of the structure as on Fig. 4a showing (in dashed lines) position and orientation of the selected residues from the
superimposed structure of the native LOX-3 (PDB entry 1LNH): Gln514, Leu515, Ile572 and V566 and waters, W936 (near His513), and W901
(overlapping with A-ring of EGC).
420 SKRZYPCZAK-JANKUN et al: EGCG AND LOX
1
OH OH
O
OOH
OH
RO
R
3'
4'
5'
6'
2'
1'
O
OH
OH
OR1
R =
OH
OH
OH
O
R1=
O
OH
OH
OH
OH
OH
OH
O
O
OH
OH
O
O
OH
OH
OH
OH
OH
OH
O
O
2
O
OH
OH
OR1
OH
OH
OH
O
OH
OH
OR1
OH
OH
OH
3
HOOC
HOOC O
OH
OH
OH
OR1
4
HOOC
HOOC
O
OH
OH
OH
OR1
5
OH
OH
OH
OH
OH
6
O
OH
OH
O
OH
O
O
7
O+
OH
OH
O
OH
O
O
8
Fig. 5. Schematic representation of different products of EGCG oxidation.
Table II. Inhibition of lipoxygenase by different catechins.
Enzyme Inhibitor Inhibitor Enzyme Reference
conc. (µM) activity
______________________________________________________
soy LOX EGCG, EGC, 10-20 50% (20)a
(mix) ECG
LOX EC 140 50% (25)b
LOX ECG 18 50% (25)b
LOX EGC 21 50% (25)b
soy LOX-3 EGC 10 45% (43) c
soy LOX-3 EGCG 1 59% (43) c
soy LOX-3 quercetin <3 52% (43) c
LOX quercetin 0.1-5 50% (25)b
___________________________________________________________________________________
a The authors used soybean lipoxydase type V from Sigma, that is
a mixture of LOX isozymes.
b This source does not specify the type of enzyme and probably
concerns soybean LOX mixture of
isozymes.
c Linoleic acid was mixed with inhibitor and LOX-3 was added to
that mixture (Skrzypczak-Jankun E, et al, 93rd Annual Meeting,
AACR, San francisco, abs.205, 2002).
______________________________________________________
The authors propose mechanism of EGCG radical oxidation
leading to gallate-CH-(OH)2 – 6, and anthocyanin-like
compounds – 7, 8 (37). Hollman and Katan (26) provide
numerous examples of flavonoids metabolites obtained from
body tissues and colon, urine and blood analysis, and the
tissue and feces collected from rats. Analysis of blood and
urine samples from humans after consumption of green tea
indicates that EGCG can be reduced to EGC in plasma, its
bioavailability is lower than EGC, and it is not detectable in
urine. It has been found that different catechins get degraded
to the small molecules consisting of only one aromatic ring,
having at least one –OH at C3 (could be methoxy) or two at
C3, C4, and substituted at C1 that gives a derivative of
valerolactone, propionic, acetic, cinnamic or simply benzoic
acid. The findings listed above indicate opening of C-ring and
farther degradation of either ring A or B. Interestingly gallate
moiety is not present among them indicating that 3’,4’,5’-
trihydroxyphenyl gets eliminated.
Interpretation of results. The medium to which EGCG was
added, was at pH~5.3 and consisted of citrate–phosphate
buffer, Tris HCl, PEG 8000 and NaN3, with protein at 2.5
mg/ml, at the aerobic conditions under atmospheric pressure
and at 23ºC. Since EGCG may not be stable in such
environment (18) all our measurement were done after >20
hours. X-ray data collection took several hours for each
crystal and formation of free radicals, including hydroxyl
INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 12: 415-422, 2003 421
radicals is unavoidable in this experimental method because it is
a normal ‘side effect’ of crystal damage caused by radiation.
This is almost never mentioned (because it is obvious), but one
should keep it in mind when interpreting results for the
biological system that contains 50% of water and might be
vulnerable to the stimulating effect of free radicals. The catechin
provided by Dr. Juneja was traced to the production lot number
and its analysis to prove that it was 98.0% of EGCG, 0.33% of
gallic acid and 1.67% of moisture. Decomposition in a sealed
container was ruled out and so whatever degradation occurred
was a result of the experimental conditions. The most likely
explanation could be the Fenton reaction initiated by hydroxyl
radical and catalyzed by enzyme. As always for such data and
resolution, a crystallographer cannot be sure about occupancy
and it is possible that in our model the occupancy of iron and
catechin could be misjudged. These two variables are correlated,
and the elevated B(Fe) = 32 Å2 and Bavg= 48 Å2 for EGC
indicate that the occupancy = 1 is somewhat overestimated.
Catechins could chelate iron and some of the enzyme cofactor
might be washed out this way. Estimation of catechin occupancy
in the complex is not straightforward either. Studies by
microcalorymetric titration of soybean LOX-3 and LOX-1 with
4-nitrocatechol and 3,4-dihydroxybenzonitrile have proven that
the number of the inhibitor molecules engaged in the complex
with LOX depends on isozyme (LOX-1 vs. LOX-3), and within
the same isozyme, on the oxidation state of iron (38, 39). The
measurements gave the stechiometry 1.3, 0.4, 0.5, 0.1 for 4-
nitrocatechol, and 1.5, 0.2, 0.5, (data for LOX-1Fe(II) not
available), for 3,4-dihydroxybenzonitrile. In general LOX-3 with
Fe(III) binds 1 or 2 molecules of the inhibitor, and at Fe(II) 0 or
1, while LOX-1 binds 0 or 1 in both cases but with occupancy
less than a half of that observed for LOX-3. Quercetin under the
same circumstances (like X-ray analysis for LOX with EGCG)
undergoes biodegradation to 3,4-dihydroxybenzoic acid (PDB
entry 1N8Q). Comparison of EGC with quercetin (Fig. 2) shows
the difference in the ring B and 5’-position. Quercetin has a
conjugated double bond system that like in curcumin (27) might
be a ‘substrate’ for peroxidase activity of lipoxygenase,
especially in the presence of suitable radicals. In case of EGC
such conjugated system is not present and further degradation of
this compound would require fission of the heterocyclic ring C
or the aromatic ring A. The weak electron density at C4 and C1’
(Fig. 3a) could mean that another type of the inhibitor molecule
could be possible – namely one aromatic moiety substituted at
positions 1, 3 and 5, similar to what was described by Hollman
& Katan (26). In that case the part of the electron density
assigned to the ring B in EGC model, could be occupied by
Gln514. This residue is a part of the hydrogen bonding network
around the active site, but gets shifted and disordered when
withdrawn by the inhibitor that propagates into this channel
(compare with PDB entries 1IK3, 1LNH) – Fig. 3b. This has to
remain a hypothesis until proven by a chemical analysis. The
compounds 7 and 8 from Fig. 5 can be ruled out because atoms
C2 and C3 of the ring C in this structure are obviously of sp3
hybridization. As for keto-enol forms of the ring B it cannot be
describe with certainty at this resolution but the contacts with
hydrophobic residues suggest that some keto-enol equilibrium
can be present there.
Kinetic studies of LOXs inhibition show that the same
inhibitor could show dramatically different results for enzymes
from different sources and different isozymes from the same
source (40, 41). Rabbit 15-LOX treated with ebselen shows
noncompetitive inactivation for Fe(II) enzyme and competitive
for Fe(III) catalytically active state. Results for LOX-3 active
form for quercetin, EGCG and EGC could be described as
competitive mechanism of inhibition but the Lineweaver-Burke
plots can be fitted to linear function to 1/{S} ~ 0.003 µM-1 and
deviate from linearity at the higher values. The deviation
increases with the growing concentration of inhibitor and in
overall indicates that the data might be much better fitted to a
non-linear function (Skrzypczak-Jankun E, et al, 93rd Annual
Meeting AACR, San Francisco, abs. 205, 2002). Comparison of
IC values cited in the literature could be deceptive because very
often they do not refer to a single enzyme and in case of soyben
LOX might be for the mixture of several isozymes (Table II),
which could be misleading. Kulkarni in his detailed review
publications gives numerous examples of co-oxidative activity of
LOX that he describes as a versatile biocatalyst for
biotransformation of endobiotics and xenobiotics (34, 42).
Therefore one may assume that the complicated pattern of LOXs
kinetics depicts not a simple inhibition but a multi(?)step
reaction of the inhibitor and its metabolites biotransformation
catalyzed by this enzyme.
Conclusions. X-ray analysis of soybean lipoxygenase-3 crystals
soaked with EGCG shows the molecular complex of LOX-3 with
(-)-epigallocatechin molecule. This finding provides another
evidence for the degradation of catechin in presence of this
enzyme. It also provides another piece of information and
contribution into understanding of an intriguing puzzle of the
drugs metabolism, and the rule of this ubiquitous enzyme in
this process.
Acknowledgements
This work was supported in part by grant from American
Diagnostica Inc., Greenwich, CT, USA and the authors thank Dr.
Richard Hart for his unfailing support. ESJ thanks Dr. Chi-Tang
Ho for helpful remarks, Robert Kernstock for performing the
kinetic measurements and Dr. O.Ya. Borbulevych for help with
drawings.
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