Characterization of xenobiotic reductase A (XenA): Study of active site residues, substrate spectrum and stability

Article abstract of DOI:10.1039/c0cc02354j

Xenobiotic reductase A (XenA) has broad catalytic activity and reduces various α,β-unsaturated and nitro compounds with moderate to excellent stereoselectivity. Single mutants C25G and C25V are able to reduce nitrobenzene, a non-active substrate for the wild type, to produce aniline. Total turnover is dominated by chemical rather than thermal instability. The Royal Society of Chemistry 2010.

Full text of DOI:10.1039/c0cc02354j

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Characterization of xenobiotic reductase A (XenA): study of active site
residues, substrate spectrum and stabilitywz
´
Yanto Yanto, Hua-Hsiang Yu, Melanie Hall and Andreas S. Bommarius*
a
a
a
ab
Received 4th July 2010, Accepted 1st October 2010
DOI: 10.1039/c0cc02354j
Xenobiotic reductase A (XenA) has broad catalytic activity and
reduces various a,b-unsaturated and nitro compounds with
moderate to excellent stereoselectivity. Single mutants C25G
and C25V are able to reduce nitrobenzene, a non-active
substrate for the wild type, to produce aniline. Total turnover
is dominated by chemical rather than thermal instability.
and C26G changed the enzyme stereoselectivity in the reduction
of a,b-unsaturated ketones.8 Overall, only few mutation studies
have been published for OYEs8b,9 to improve their catalytic
properties and we wish to report efforts toward this goal.
In this work, the substrate spectrum, stereoselectivity, and
stability of XenA were characterized. The selection of mutants
was based on the results of iterative saturation mutagenesis
(ISM) around the binding pocket of YqjM, which implied
both residues C25 and A59 in substrate binding.8b The Quik-
Changes mutagenesis protocol was used to generate C25G,
C25V, and A59V mutants. We analyzed wild type XenA (WT)
and the effect of mutations on thermal and chemical stability,
enzyme activity, and regio- and stereoselectivity with various
a,b-unsaturated compounds and nitro compounds (Table 2).
Constant cofactor concentration was maintained using a
recycling system with glucose dehydrogenase (GDH) in a
conversion study (Scheme 1). The reaction products were
extracted and analyzed by gas chromatography to determine
substrate conversion and product enantiopurity. Enzyme
activities were monitored on a spectrophotometer for specific
The Old Yellow Enzyme (OYE) family is a class of flavin-
dependent enzymes that catalyze the stereoselective reduction
of activated CQC bonds at the expense of nicotinamide
cofactors. This asymmetric bioreduction creates up to two
new stereogenic centers and thus has gained increasing interest
as a new tool in chiral intermediates synthesis.1 Members of
the OYE family were reported to catalyze the bioreduction
of a variety of different substrates such as a,b-unsaturated
aldehydes, ketones, imides, nitro groups, nitriles, carboxylic
acids, nitro esters, and nitro aromatics.2
The catalytic activity of xenobiotic reductase A (XenA)
from Pseudomonas putida was first reported for the reduction
of nitroglycerin (reductive denitration), TNT (reduction of nitro
group) and 2-cyclohexen-1-one (reduction of olefinic bond),3
and later for nitroreductase activity for cyclotrimethylene-
trinitramine (RDX, Research Department Explosive) and a
series of nitroaromatic and nitramine explosive compounds.4
This FMN containing enzyme was also shown to display
broad enoate reductase activity over various a,b-unsaturated
carbonyl compounds such as citral, ketoisophorone, maleimide,5
8-hydroxycoumarin, and coumarin.6 XenA shares a generally low
amino acid sequence identity and similarity with other OYE
enzymes and structural variation was observed in the active site
of XenA compared to the OYE family. A recently published
study focused on XenA active site residue C25, which replaces the
highly conserved threonine residue in OYE enzymes. This impor-
tant residue interacts with FMN and was shown to modulate the
flavin reduction potential and thus influence substrate binding in
XenA.7 An analogous residue C26 in YqjM from Bacillus subtilis
(OYE enzyme that shares the highest sequence identity and
similarity with XenA: 40.1% identity and 53.8% similarity,
respectively) was shown to act as a redox sensor that controls
the reduction potential of FMN in YqjM, and mutation C26D
Scheme 1 Asymmetric trans-bioreduction of CQC bonds coupling
XenA with cofactor recycling system through half-oxidative and
half-reductive reaction (EWG
= electron withdrawing group,
GDH = glucose dehydrogenase, * = center of chirality).
Table 1 XenA specific activity and stability study
XenA Constructs
Substrate
WT
C25G
C25V
A59V
Specific Activity [U/mg]
1-Nitrocyclohexene 5.87
0.86
0.80
0.21
0.16
0.35
0.47
0.70
0.07
0.16
0.05
0.02
0.02
2-Cyclohexenone
Ketoisophorone
2-Methylfuran
3.84
1.34
0.15
a School of Chemical and Biomolecular Engineering,
Parker H. Petit Institute of Bioengineering and Biosciences,
Georgia Institute of Technology, 315 Ferst Drive, Atlanta, GA,
USA. E-mail: andreas.bommarius@chbe.gatech.edu;
Fax: (+1) 404-894-2291; Tel: (+1) 404-385-1334
b School of Chemistry and Biochemistry, Georgia Institute of
Technology, 901 Atlantic Drive, Atlanta, GA, USA
w This article is part of the ‘Enzymes and Proteins’ web-theme issue for
ChemComm.
Tm/1C
50.4
63
0.0119
1959
52.7
49.5
0.0136
1073
48.0
44.7
0.0155
280
63.5
t1/2 at 37 1C/h
>112
o0.0062
38
kd,obs/hꢀ1
TTNchem
TTNthermal (37 1C)
8.3 ꢁ 105 1.4 ꢁ 105 7.2 ꢁ 104 1.9 ꢁ 104
Specific activity assay: 200 mM phosphate buffer pH 7.5, 1 mM
substrate, 0.2 mM NADPH, 10 mg mLꢀ1 (0.25 mM) enzyme, 37 1C.
t1/2 assay: Enzymes incubated at 37 1C and residual activity measured
with 2-cyclohexenone at different time points.
z Electronic supplementary information (ESI) available: Experimental
details and product characterization. See DOI: 10.1039/c0cc02354j
c
This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 8809–8811 8809

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activity, half-life at 37 1C, and total turnover number (TTN)
studies (Table 1) (see ESI for detailed proceduresz).
t1/2 of 44.7 h. (Table 1) Surprisingly, A59V mutant showed major
improvement in Tm to 63.5 1C and consequently higher t1/2 at
37 1C. XenA and YqjM were proposed to be stabilized through
simple salt bridges involving single pairs of charged amino
acids.12 However, the single A59V mutation showed significant
influence on thermostability (although as an uncharged residue,
it cannot form any salt bridge), while the C25 residue did not
show significant influence on thermostability.
The enzymatic activity of XenA and mutants was first
investigated on four different substrates: 1-nitrocyclohexene,
2-cyclohexenone, ketoisophorone, and 2-methylfuran. WT
showed highest specific activity compared to all other mutants
on the four compounds. Both C25 mutants showed signifi-
cantly lower reactivity for all substrates with o 1 U mgꢀ1
specific activities observed. Mutation on the C25 residue was
previously shown to strongly affect both oxidative half reac-
tion towards the substrate and reductive half reaction towards
the cofactor.7 Our results confirmed the importance of C25 in
the overall reaction as shown by the much lower specific
activity of the mutants compared to the wild type. Mutation
of A59V had a dramatic effect on XenA, as it almost led to
disappearance of any enoate reductivity. We also observed
uncompetitive substrate inhibition with 2-cyclohexenone while
The total turnover number (TTN) is an indicator of life-
time biocatalyst productivity and is defined as the maximum
number of molecules of substrate that an enzyme active site
13
can convert over its life cycle. The chemical TTN (TTNchem
)
was experimentally determined by measuring the maximum
amount of substrate 2-cyclohexenone that can be reduced
with a set amount of purified enzyme before the enzyme is
chemically deactivated. The thermal TTN (TTNthermal) was
based on the thermal deactivation of enzyme, calculated from
the ratio kcat/kd,obs, valid for enzymes with 1st order thermal
deactivation,14 such as XenA (see ESI for detailed procedures
and calculationsz). The WT showed the highest TTN values
compared to all other variants. Mutant A59V showed the
lowest TTNchem of 38 and TTNthermal of 1.9 ꢁ 104. Overall, the
results showed that XenA enzyme reactivity was dominated by
chemical rather than thermal deactivation, and that single
point mutations on C25 and A59 led to significant decrease
in TTN. The cause for the chemical turnover-dependent
deactivation is under investigation. Turnover-driven stability
limits have also been observed in D-amino acid oxidase in
connection with the mutation of the C108 residue.15
inhibition has never been observed with XenA before.6,7
A
non-linear fit of the Michaelis–Menten equation yielded kcat of
25.7 sꢀ1, KM of 0.19 mM, and inhibition constant KI of
0.15 mM (see ESIz).
Due to its relatively high sequence identity with YqjM,
XenA belongs to the same subclass of OYEs that has been
proposed to contain ‘‘thermostable-like’’ enzymes along with
recently identified thermostable OYE from T. pseudethanolicus,10
and chromate reductase from the thermophile T. scotoductus.11
One characteristic of this subclass is the conservation of C25,
which was observed in both XenA and YqjM. We investigated
the effect of C25 mutation on the thermal stability by measuring
both the melting temperature (Tm) and the half-life (t1/2) at 37 1C
(see ESIz). The Tm of WT was determined to be 50.4 1C with t1/2
of 58.2 h at 37 1C. C25G displayed a slightly higher Tm (52.7 1C)
but a lower t1/2 (50.9 h), whereas C25V showed opposite
behavior with Tm (reduced to 48.0 1C) and a significantly lower
The conversion study of XenA was performed over 12 h
with multiple sets of substrates: a,b-unsaturated carbonyl
compounds (1–5), a,b-unsaturated nitro compounds (6–9),
nitro aromatic compounds (10–11), and nitrofuran-like
substrates (12–14). This is the first study where nitrofuran
Table 2 Biocatalytic conversion of various a,b-unsaturated carbonyl, nitrostyrene, nitroaromatic, and nitrofuran substrates
XenA WT
Conv. (%)
C25G
C25V
A59V
e.e. (%)
Conv. (%)
e.e. (%)
—
59.9 (R)
>99.9 (R)
37.7 (S)
99.5 (R)
72.9 (S)
—
—
—
—
—
—
—
—
Conv. (%)
e.e. (%)
Conv. (%)
e.e. (%)
Substrate
1. 2-Cyclohexenone
96.4
19.7
0.4
98.9
8.1
—
99.1
12.9
6.4
21.5
32.4
0.9
—
86.1 (R)
n.d
14.9 (S)
99.3 (R)
2.7 (S)
—
—
—
—
—
7.1
0.5
—
—
—
2. 2-Methyl-2-cyclopentenone
3. 3-Methyl-2-cyclohexenone
4. Ketoisophorone
78.5 (R)
n.dc
2.25 (S)
99.5 (R)
69.9 (S)
—
—
—
—
—
No conv.
3.8
18.4
54.1
33.5
33.3
15.6
36.1
29.3
59.9
54.3
0.7
87.7
18.5
11.7
No conv.
2.7
25.6 (S)
68.0 (R)
98 (S)
5. Citral
6. 1-Nitro-2-phenylpropenea
7. trans-b-Nitrostyrene
8. trans-b-Methyl-b-nitrostyrene
9. 1-Nitrocyclohexene
10. 2,4-Dinitrotoluene
11. Nitrobenzeneb
7.1
6.3
5.0
10.1
13.6
91.2
60.2
1.1
47.0
17.4
8.3
—
—
—
—
—
—
—
—
3.6
No conv.
6.6
No conv.
5.8
22.9
53.2
23.2
5.9
1.3
7.0
12. 2-Nitrofuran
13. 2-Methyl-5-nitrofuran
14. 2-(2-Nitrovinyl)furan
—
—
—
—
—
—
5.8
7.9
9.3
5.8
Conversion assay: 200 mM phosphate buffer pH 7.5, 5 mM substrate, 20 mg mLꢀ1 (0.5 mM) enzyme, 0.2 mM NADP+, 20 mM glucose, 10 U mLꢀ1
GDH.a The conversion of 6 showed side products other than 1-nitro-2-phenyl-propane, which are possibly the corresponding carbonyl
compounds similar to Nef-reaction products.18 b Conversion of 11 with C25G and C25V produced nitrosobenzene, aniline, and azobenzene.
c
n.d: not determined.
c
8810 Chem. Commun., 2010, 46, 8809–8811
This journal is The Royal Society of Chemistry 2010

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type substrates are tested with true OYE enzymes. The WT
showed high conversion efficiency in CQC reduction of sub-
strates 2, 4, and 9; and modest conversion for substrates 2, 10,
and 12. The WT also showed moderate to excellent stereo-
selectivity for reduction of 2 (78.5% R), 5 (99.5% R), and 6
(69.9% S), although a racemic product was obtained in the
reduction of 4 (2.25% S).16 Overall, the WT seems to show
preference for cyclic compounds (except 2 and 3). Mutation
C25G improved the conversion and stereoselectivity with
various substrates compared to the wild type: conversion of
3 was increased to 6.4% from 0.4% with product e.e > 99.9%
(R); conversion of 5 was increased fourfold with similar
product e.e; conversion of 6 was also improved by 4-fold to
33.3% with slightly improved product e.e. Most importantly,
one single mutation was able to significantly increase the
conversion of 11 (5 mM) from 1.1% to 54.3% to produce
nitrosobenzene (0.18 mM), aniline (0.15 mM), and azobenzene
(2.6 mM). C25V mutant showed higher conversion efficiency
of substrates 2 and 5 with similar or slightly enhanced product
e.e; conversion of substrate 6 was slightly improved, the
product was however nearly racemic with e.e of 2.7% (S);
C25V also catalyzed the reduction of 11 and produced similar
products to C25G mutant, including aniline. Mutant A59V
showed lower conversion for most of the tested substrates.
However, the stereoselectivity in the reduction of 6 was
significantly improved to 98% (S) while maintaining similar
conversion efficiency to WT. Upon this result, we analyzed the
reduction of 6 with the double mutant C25G/A59V. However,
C25G/A59V showed negligible activity, even with 2-cyclo-
hexenone as substrate. All mutants showed highly reduced
enzymatic activity towards the reduction of nitrofurans
(12–14), except for A59V that retained activity towards 13.
Both single mutants C25G and C25V surprisingly catalyzed
the reduction of 11 to produce aniline, while WT was nearly
inactive. A similar reduction pattern was also reported for
NRSal from S. typhimurium.17 We propose the removal of the
C25 residue changes the binding conformation of 11 in the
catalytic pocket, thus allowing the subsequent reduction of
the nitro group via a two-electron reduction pathway and the
formation of aniline. Traces of phenylhydroxylamine were
also observed alongside the enzyme-catalyzed formation of
aniline. Formation of azobenzene as side product was detected
likely due to the chemical condensation reaction between
nitrosobenzene and phenylhydroxylamine.19
melting temperature, and TTN as an indicator for biocatalyst
productivity. The results showed the effect of chemical
instability is more significant than thermal instability.
Notes and references
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In conclusion, we have characterized the substrate spectrum
of WT XenA and mutants. The enzyme showed a high degree
of conversion and marked stereoselectivity and was able to
reduce CQC bonds as well as nitro compounds. Mutations on
C25 interestingly showed significantly stronger nitroreductase
activity to produce the corresponding amino product. We also
studied both chemical and thermal enzyme stability,
c
This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 8809–8811 8811