The thiamine-dependent enzyme of the vitamin K biosynthesis catalyzes reductive C-N bond ligation between nitroarenes and α-ketoacids

Article abstract of DOI:10.1007/s11426-012-4792-6

The thiamine-dependent enzyme (1R, 2S, 5S, 6S)-2-succinyl-5-enolpyruvyl-6- hydroxyl-3-cyclohexene-1-carboxylate (SEPHCHC) synthase, also known as MenD, catalyzes a Stetter-like reaction in the biosynthesis of vitamin K. It is found to catalyze a novel reductive C-N bond ligation reaction between nitroarenes and α-ketoacids to form N-hydroxamates. This reaction likely proceeds through an enzyme-mediated, slow two-electron reduction of the nitroalkanes to form a nitroso intermediate, which serves as the electrophilic acceptor of the ketoacid-derived acyl anion. The involvement of the nitroso intermediate is supported by the fact that similar N-hydroxamates are readily formed at a much higher rate when nitroso compounds replace the nitro substrates in the chemoenzymatic reactions. These results demonstrate that the thiamine-dependent enzyme is able to catalyze novel, nonnative reactions that may find new chemoenzymatic applications.

Full text of DOI:10.1007/s11426-012-4792-6

SCIENCE CHINA
Chemistry
March 2013 Vol.56 No.3: 312–320
doi: 10.1007/s11426-012-4792-6
• ARTICLES •
· SPECIAL TOPIC · The Frontiers of Chemical Biology and Synthesis
The thiamine-dependent enzyme of the vitamin K biosynthesis
catalyzes reductive C–N bond ligation between nitroarenes and
-ketoacids
CHEN MinJiao, JIANG Ming & GUO ZhiHong*
Department of Chemistry and State Key Laboratory for Molecular Neuroscience, The Hong Kong University of Science and Technology,
Clear Water Bay, Kowloon, Hong Kong SAR, China
Received September 25, 2012; accepted October 30, 2012; published online December 4, 2012
The thiamine-dependent enzyme (1R, 2S, 5S, 6S)-2-succinyl-5-enolpyruvyl-6-hydroxyl-3-cyclohexene-1-carboxylate (SEPHCHC)
synthase, also known as MenD, catalyzes a Stetter-like reaction in the biosynthesis of vitamin K. It is found to catalyze a novel
reductive CN bond ligation reaction between nitroarenes and -ketoacids to form N-hydroxamates. This reaction likely pro-
ceeds through an enzyme-mediated, slow two-electron reduction of the nitroalkanes to form a nitroso intermediate, which
serves as the electrophilic acceptor of the ketoacid-derived acyl anion. The involvement of the nitroso intermediate is support-
ed by the fact that similar N-hydroxamates are readily formed at a much higher rate when nitroso compounds replace the nitro
substrates in the chemoenzymatic reactions. These results demonstrate that the thiamine-dependent enzyme is able to catalyze
novel, nonnative reactions that may find new chemoenzymatic applications.
chemoenzymatic synthesis, N-hydroxamates, thiamine-dependent enzymes, SEPHCHC synthase, vitamin K biosynthesis
reactions in microorganisms, plants, and mammals in di-
verse biological processes [4].
1 Introduction
The carbon-carbon bond forming ThDP-dependent en-
zymes are attractive targets for biotechnological exploita-
tion as versatile biocatalysts for chemoenzymatic synthesis
[5, 6]. Pyruvate decarboxylase (PDC) and benzoylformate
decarboxylase (BFD) are two successful examples of such
efforts, although both enzymes do not catalyze carbon-
carbon bond formation in their physiological activities. Re-
markably, they tolerate a wide range of structural variations
in their substrates, allowing access to a large number of
enantiopure products. For decades, yeast PDC has been
used to prepare the (R)-phenylacetyl carbinol precursor by
fermentation for industrial production of (1R, 2S)-ephedrine
[7, 8], a versatile chiral synthon in organic synthesis [9, 10]
and an agonist of noradrenaline receptors with clinical ap-
plications [11]. In addition, transketolase is another suc-
cessful biocatalyst in chemoenzymatic synthesis [12]. It
Thiamine diphosphate (ThDP)-dependent enzymes typically
convert an electron-deficient carbonyl substrate into a nu-
cleophile in their catalysis. An important step in their catal-
ysis is activation of the ThDP coenzyme through deprotona-
tion of C2-proton of the thiazolium ring. The resulting ylide
then attacks an -ketoacid substrate to generate an enamine
carbanion intermediate or acyl anion after decarboxylation
(Figure 1) [1–3]. Subsequently, the electron-rich intermedi-
ate undergoes rearrangement, oxidation, or nucleophilic
addition to electrophilic acceptors to form new carbon-
carbon bonds. Over the last several decades, a large number
of ThDP-dependent enzymes have been discovered to form
a large enzyme superfamily catalyzing a broad range of
*Corresponding author (email: chguo@ust.hk)
© Science China Press and Springer-Verlag Berlin Heidelberg 2012
chem.scichina.com www.springerlink.com

Chen MJ, et al. Sci China Chem March (2013) Vol.56 No.3
313
Figure 1 The proposed catalytic mechanism of SEPHCHC synthase. SEPHCHC: (1R, 2S, 5S, 6S)-2-succinyl-5-enolpyruvyl-6-hydroxyl-3-cyclohexene-
1-carboxylate; Pi: phosphate group.
transfers a glycoaldehyde unit from either a donor ketose or
hydroxypyruvate to an -hydroxyaldehyde acceptor and has
been used to synthesize a number of bioactive molecules
[13–16]. It has also been suggested for use as an alternative
to the versatile aldolase for synthesis of a number of natural
products [17].
of the nitroso intermediate is supported by the formation of
similar N-hydroxamates from nitroso compounds and
-ketoacids. The finding of this CN bond ligation reaction
has provided new opportunities to exploit the
ThDP-dependent enzymes in chemoenzymatic synthesis.
Recently, a new ThDP-dependent enzyme called MenD
2 Materials and methods
or
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-
cyclohexene-1-carboxylate (SEPHCHC) synthase has been
characterized in the biosynthesis of menaquinone (vitamin
K2) [18, 19]. Different from all other carbon-carbon bond
forming ThDP-dependent enzymes that use carbonyl com-
pounds as the electrophilic acceptor, this enzyme uses iso-
chorismate, an ,-unsaturated carboxylate, as the electro-
philic acceptor of the acyl anion generated from
2-oxo-glutarate. Structural and mechanistic studies have
confirmed that the enzyme contains a conserved glutamate
to activate the ThDP cofactor as a thiazolium ylide, similar
to other ThDP-dependent enzymes [20, 21]. Its catalytic
mechanism is proposed as shown in Figure 1, which is sim-
ilar to that of other ThDP-dependent enzymes except for the
nucleophilic 1,4-Michael addition. The finding of this en-
zyme has provided new opportunity for exploitation of the
ThDP-enzymes in chemoenzymatic syntheses. Indeed, the
MenD orthologue from E. coli has been shown to use un-
natural carbonyl compounds as the electrophilic acceptor of
the acyl anion to form stereospecific products [22].
2.1 General information
2-Oxo-glutarate, thiamine diphosphate (ThDP), 4-nitrocin-
namic acid (97%, predominantly in trans-configuration),
isopropyl β-D-thiogalactopyranoside (IPTG), buffers, and
salts were purchased from Sigma-Aldrich. Other chemicals
and biochemicals were obtained from commercial sources
with the highest possible quality and used without further
treatment, unless stated otherwise. Protein chromatography
was performed on a Bio-Rad Bio Logic HR Workstation in
a refrigerated chamber. HPLC analysis and purification
were carried out using a Waters 600E system with Model
2487 dual  absorbance detector. UV-vis absorbance was
measured using a Perkin-Elmer Lambda 900 UV/vis/NIR
spectrometer. The molecular weight of the products from
enzymatic reactions was determined by high resolution
electron-spray ionization (ESI) mass spectroscopy. Nuclear
magnetic resonance (NMR) spectra were taken on 400 MHz
Bruker spectrometers.
In attempt to use MenD for chemoenzymatic synthesis of
-oxo carboxylic acids, the enzyme was tested for its activi-
ty towards unnatural , -unsaturated carboxylates such as
4-nitrocinnamic acid. Unfortunately, the enzyme was found
to exhibit a high substrate specificity and no expected 1,
4-addition product was formed. Quite unexpectedly,
N-hydroxamates were formed from nitro-containing com-
pounds and -ketoacids through a reductive C–N bond liga-
tion likely involving a nitro intermediate. The involvement
2.2
bsMenD
Expression, purification and activity test of
The MenD orthologue from Bacillus subtilis (bsMenD) was
expressed using a recombinant plasmid containing the target
gene in a modified version of the Novagen pET15b vector,
in which the thrombin cleavage site is replaced by a Tobac-
co Etch Virus (TEV) protease site. The recombinant plas-

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Chen MJ, et al. Sci China Chem March (2013) Vol.56 No.3
mid was a generous gift from professor William N. Hunter
of the University of Dundee [21]. To obtain the enzyme, the
plasmid was introduced into the host cell E. coli BL21 (DE3)
and the gene product was overexpressed in Luria broth con-
taining 0.5 mM IPTG at 16 °C for 18 h. The expressed en-
zyme was then purified from the crude extract to greater
than 95% purity by a 5.0 mL HiTrap chelating HP column
(GE Healthcare). The purified protein was quantified by a
Coomassie Blue protein assay kit (Pierce) and stored in
phosphate buffered saline (pH 7.4) containing 10% glycerol
at 20 °C until use.
For determination of the bsMenD activity, EntC obtained
in previous studies [23, 24] was used to prepare isochoris-
mate from chorismate, which was isolated from an engi-
neered bacterial strain using a reported procedure [25]. The
enzyme activity was either determined by monitoring the
decrease of absorbance at 278 nm due to the consumption of
isochorismate [18] or by monitoring the absorbance increase
at 293 nm in an assay using the (1R,6R)-2-succinyl-6-
hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC) syn-
thase MenH [26, 27] as the coupling enzyme. The kinetic
parameters Vmax and KM were determined in the presence of
saturating concentration of the cofactors and one of the sub-
strates. All kinetic assays were carried out in 50 mM Tris
buffer (pH 7.8) containing 50 mM NaCl and 20 mM MgCl2
at 23 °C.
wavelength of the reactant or the product. The reaction
mixture was analyzed on an Nova-Pak C18 reverse-phase
column (4 m particle size, 3.9  150 mm) using a linear
gradient from 100% solution A to 100% solution B over 60
min at a flow rate of 1 mL/min, where solution A was 0.1%
formic acid water solution and solution B was 100% ace-
tonitrile. Control reactions without enzyme were treated in
parallel and analyzed under the same conditions. To quan-
tify the product formed in the enzymic reactions,
2-nitrobenzoic acid was added into the mixture to a final
concentration of 100 µM and was used as an internal stand-
ard in the HPLC analyses. Integration areas of the elution
peaks of the chromogenic substrate (such as 4-nitroci-
annamic acid) and the internal control with known concen-
trations were calibrated to determine conversion rate of the
substrate at different reaction times.
2.4 Preparation of N-[4-(2′-carboxy-(E)-vinyl)phenyl]
succinyl hydroxamate (1) and other turnover products
Enzymatic preparation of product 1 was performed in 5 mL
of 50 mM Tris buffer (pH 7.8) containing 2 mM 4-nitro-
cinnamic acid, 4 mM 2-oxo-glutarate, 10 mM MgCl2, 200
µM ThDP and 20 µM MenD. 2-oxo-Glutarate was replen-
ished to a final concentration of 4 mM every 6 h during the
reaction, assuming that the previously added substrate was
completely consumed. The reaction mixture was incubated
at room temperature for 2 d (> 60% consumption of
4-nitrocinnamic acid) and quenched by addition of HCl to
pH ~1. The products were extracted with ethyl acetate (4 
5 mL). The organic extracts were combined and the organic
solvent was removed under reduced pressure. The residue
was re-suspended in 1 mL mixture of water and acetonitrile
(1:1) and subjected to purification by a semi-preparative
HPLC column. The product fractions were pooled, concen-
trated under reduced pressure to remove acetonitrile, and
then lyophilized to obtain the product with 60% yield
(4-nitrocinnamic acid as the limiting agent). Other turnover
products of the nonphysiological reactions of bsMenD were
similarly prepared.
2.3 bsMenD activity with nonphysiological substrates
Taking 4-nitrocinnamic acid as the substitute of isochoris-
mate for an example, the reaction rate was monitored in real
time by full wavelength UV scanning. UV-vis spectra were
taken using a Perkin-Elmer Lambda 900 UV/vis/ NIR spec-
trometer. A typical reaction mixture contained 200 µM
4-nitrocinnamic acid, 2 mM 2-oxo-glutarate, 200 µM ThDP
in 50 mM Tris buffer (pH 7.8) supplemented with 50 mM
NaCl and 20 mM MgCl2 at room temperature. The reaction
was initiated by adding the enzyme to a varied concentra-
tion. Since 2-oxo-glutarate was continuously consumed to
form 2-hydroxy carbonyl products as shown previously [22],
fresh substrate was added to an equivalent final concentra-
tion of 2 mM every 2 h during the reaction. This replenish-
ment of 2-oxo-glutarate was also performed in activity tests
using other electrophilic acceptors as substitute of the iso-
chorismate substrate. In tests where the 2-oxo-glutarate sub-
strate was also replaced, concentration of the substituting
ketoacid was not fixed but varied in a range from 200 µM to
10 mM while keeping other reaction components un-
changed.
3 Results and discussion
3.1 Reductive C–N bond ligation between 4-nitrocin-
namic acid and 2-oxo-glutarate
bsMenD was overexpressed to a very high level in E. coli
and a yield of 100 mg protein per 1 liter shake-flask culture
was readily attainable. The purified enzyme was homoge-
nous after metal chelating column chromatography and ex-
hibited an activity consistent with results from previous
characterization of the enzyme [21]. To explore the sub-
strate tolerance of the enzyme, cinnamic acid and its deriva-
tives were used as substitute for the isochorismate substrate
in the bsMenD-catalyzed reaction, which was monitored by
In addition to the spectroscopic monitoring, the reaction
products were also monitored and quantified by HPLC. To
do this, the reaction was stopped by removing the enzyme
through ultrafiltration and HPLC analysis was performed on
a Waters 600E system with Model 2487 dual  absorbance
detector set at the characteristic ultraviolet absorption

Chen MJ, et al. Sci China Chem March (2013) Vol.56 No.3
315
ultraviolet spectroscopy and HPLC analysis. Interestingly,
when 4-nitrocinnamic acid was used in the reaction, the
electrophilic compound was obviously modified. As shown
in Figure 2(a), the characteristic absorption of 4-nitrocin-
namic acid at 312 nm gradually decreased over time while a
new absorption peak at 270 nm emerged and its intensity
increased as the reaction proceeded. Reverse-phase HPLC
analysis of the enzymic reaction mixture confirmed that
4-nitrocinnamic acid was indeed transformed into a new
product with a higher polarity (Figure 2(b)) and the conver-
sion was complete (> 99%) after 48 hours of incubation at
room temperature. However, no spectroscopic or HPLC
changes were observed when cinnamic acid, 4-bromo-
cinnamic acid, or 4-methyl-cinnamic acid was used in the
enzymic reaction (data not shown). In all these reactions,
2-oxo-glutarate undergoes benzoin-like condensation as
shown previously [22], but this reaction is not detectable by
the employed method due to the lack of ultraviolet absorp-
tion for the ketoacid substrate and the condensation product.
This ketoacid substrate was replenished as indicated in the
methods and materials to compensate for the loss of the
substrate during the reaction.
The turnover product from the bsMenD-catalyzed reac-
tion between 4-nitrocinnamic acid and 2-oxo-glutarate was
purified by HPLC from the reaction mixture for structural
determination. 1H NMR spectroscopy reveals a C=C double
bond in the product, as indicated by two olefinic protons in
trans-configuration at 7.69 (d, J = 16.0 Hz, 1H) and 6.52 (d,
J = 16.0 Hz, 1H), demonstrating that the expected 1,
4-Michael addition of the acyl anion to the electrophilic
4-nitrocinnamic acid does not occur in the reaction. The
product has a molecular formula of C13H13NO6, which has a
calculated mass of 279.0743, consistent with the major mo-
lecular ions [M + H]+ at m/z = 280.0831 and [M + Na]+ at
m/z = 302.0675 in the mass spectrometry. Using COSY,
1
HMBC and HMQC techniques, all H NMR signals of the
product are assigned as shown in Table 1. These results
show that the product is N-[4-(2′-carboxy-(E)-vinyl)phenyl]
succinyl hydroxamate (1), which is obviously formed from
reduction and nucelophilic addition at the nitro group of
4-nitrocinnamic acid. Since no reaction was observed for
cinnamic acid or its other derivatives without a nitro group,
the nitro group may be the only structural element required
for the observed reaction.
3.2 Reductive C–N bond ligation between other nitro
compounds and 2-oxo-glutarate
Inspired by the result for 4-nitrocinnamic acid, several other
nitro compounds were subjected to bsMenD-mediated reac-
tion with 2-oxo-glutarate. As shown in Table 2, 3-nitro-
benzoate, 4-nitrobenzoate and nitrobenzene indeed undergo
reductive coupling reaction with 2-oxo-glutarate to form
corresponding hydroxamates, whereas no reaction occurs
for other nitro compounds. The lower conversion rate for
nitrobenzene is probably due to its low aqueous solubility,
which may also underlie the lack of reactivity for ni-
troethane. Interestingly, all the inactive nitro compounds
contain an electron-donating group to increase the electron
density of the nitro group, thereby decreasing its oxidative
potential. These results suggest that a high oxidative poten-
tial is needed for the reductive coupling reaction.
Characterization data for N-(4-carboxyphenyl) succinyl
hydroxamate (2) from 4-nitrobenzoic acid: C11H11NO6,
calcd. 253.0586; found 254.0692 ([M + H]+) and 276.0516
([M + Na]+). H (400 MHz, CD3OD): 3.06 (2H, t, J = 6.40
Hz,), 2.71 (2H, t, J = 6.40 Hz), 7.87 (2H, d, J = 8.80 Hz),
and 8.07 (2H, d, J = 8.80 Hz).
Characterization data for N-(3-carboxyphenyl) succinyl
hydroxamate (3) from 3-nitrobenzoic acid: C10H11NO6,
calcd. 253.0586; found 254.0693 ([M + H]+) and 276.0513
([M + Na]+). H (400 MHz, CD3OD): 3.02 (2H, br, s), 2.70
Figure 2 The bsMenD-catalyzed reaction of 4-nitrocinnamic acid and
2-oxo-glutarate. (a) UV-vis spectroscopic monitoring of the reaction.
Spectra were taken every 15 min; (b) HPLC analysis of the reaction prod-
uct at different time points. The reaction was carried out at 25 C in a solu-
tion containing 65 µM 4-nitrocinnamic acid, 2 mM 2-ketoglutarate, 100
µM ThDP and 7.4 µM bsMenD in 50 mM Tris buffer (pH 7.8) supple-
mented with 50 mM NaCl and 20 mM MgSO4. Control reaction did not
contain the enzyme.

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Chen MJ, et al. Sci China Chem March (2013) Vol.56 No.3
Table 1 Assignment of the NMR signals of the product N-[4-(2′-carboxy-(E)-vinyl)phenyl]-succinyl hydroxamate (1)
δ 1H (ppm)
no proton
7.79, d, 2H,
J = 4.4 Hz
7.66, d, 2H,
J = 4.4 Hz
no proton
7.66, d, 2H,
J = 4.4 Hz
7.79, d, 2H,
J = 4.4 Hz
no proton
3.03, t, 2H
2.72, t, 2H
No proton
7.69, d, 1H,
J = 16 Hz
6.52, d, 1H,
J = 16 Hz
no proton
δ
13C (ppm)
118.9
Position
C1
COSY
HSQC
HMBC
C2
7.66
7.79
118.9
127.4
C3
C4
C5
127.4
127.9
127.4
127.9, 143.4
127.9, 143.4
7.66
7.79
127.4
118.9
C6
118.9
C7
C8
C9
28.2
27.4
173.9
2.72
3.03
28.2
27.4
173.9, 28.2
C10
C11
143.4
6.52
7.69
143.4
127.9, 169.9
C12
C13
117.5
169.9
Table 2 Summary of the bsMenD-catalyzed reductive CN bond ligation between nitro compounds and 2-oxo-glutarate a)
Substrate
Product
Conversion (%) b)
>99
>99
>70
>50
no product
no product
no product
0
0
0
no product
no product
0
0
no product
no product
0
0
a) Reaction mixture contained 100200 µM nitro compound, 2 mM 2-oxo-glutarate, ThDP 250 µM and 8.9 M MenD in 50 mM Tris buffer (pH 7.8)
supplemented with 50 mM NaCl and 10 mM MgCl2 and was incubated at 25 C for 20 h. 2-Oxo-glutarate was replenished every 2 hours; b) the conversion
rate was determined by HPLC analysis as described in the Materials and methods.

Chen MJ, et al. Sci China Chem March (2013) Vol.56 No.3
317
(2H, t, J = 6.80 Hz), 7.45–7.66 (2H, m, aromatic),
7.24–7.27 (2H, m, aromatic).
3.3 C–N bond ligation between nitroso compounds and
2-oxo-glutarate
Nitrosoarenes are structurally similar to nitroarenes and
may react with 2-oxo-glutarate in the presence of bsMenD
activity like the latter. To test this possibility, two commer-
cially available nitroarenes, nitrosobenzene and 2-nitro-
sotoluene, were used in the chemoenzymatic synthesis. The
reactions were similarly subjected to real-time spectroscopic
monitoring as well as by HPLC analysis. As shown in Fig-
ure 3(a), 2-nitrosotoluene reacted very fast with the ketoacid,
as indicated by the fast disappearance of the characteristic
absorbance of the nitroso compound at 280 nm. The reac-
tion was complete within 15 min. HPLC analysis found that
a product was formed and its amount was not changed for 2
h. Similarly, very fast reaction was observed for nitro-
sobenzene. The products of these two reactions were puri-
fied and determined to be N-(2-methylphenyl) succinyl hy-
droxamate (4) and N-phenyl succinyl hydroxamate (5), re-
spectively. The isolation yield of both products was > 60%.
Characterization data for N-(2-methylphenyl) succinyl
hydroxamate (4): C11H13NO4, calcd. 223.0845, found
224.0941 ([M + H]+) and 246.0745 ([M + Na]+); H (400
MHz, CD3OD): 2.3 (3H, s), 3.05 (2H, t, J = 6.80 Hz), 2.72
(2H, t, J = 2.80 Hz), 7.33 (4H, m, aromatic). Characteriza-
tion data for N-phenyl succinyl hydroxamate (5):
C10H14NO4, calcd. 209.0688, found 210.0708 ([M + H]+)
and 232.0607 ([M + Na]+); H (400 MHz, CD3OD): 3.04
(2H, s, br), 2.72 (2H, t, J = 6.80 Hz), 7.50–8.50 (5H, m,
aromatic).
Figure 3 The bsMenD-catalyzed reaction of 2-nitrosotoluene and 2-
oxo-glutarate. (a) Reaction of 2-nitrosotoluene and 2-ketoglutarate with
UV-vis spectra taken every 1.5 min. The reaction was carried out at 25 C
in a solution containing 100 µM 2-nitrosotoluene, 3 mM 2-ketoglutarate,
100 µM ThDP and 2.68 µM bsMenD in 50 mM Tris buffer (pH 7.8) sup-
plemented with 50 mM NaCl and 10 mM MgCl2; (b) HPLC analysis of the
reaction product at different time points. The reaction was carried out at 25
C in a solution containing 200 µM 2-nitrosotoluene, 2 mM 2-ketoglutarate,
100 µM ThDP and 268 nM bsMenD in 50 mM Tris buffer (pH 7.8) sup-
plemented with 50 mM NaCl and 10 mM MgCl2. The control reaction did
not contain bsMenD.
3.4 C–N bond ligation between nitroso compounds
with other -ketoacids
Due to the high reactivity of the nitroso compounds with
2-oxo-glutarate, we used them to explore the substrate spec-
ificity of bsMenD towards -ketoacids. The results for six
-ketoacids are shown in Table 3. The expected hydrox-
amate products were detected by HPLC only for 2-oxo-
butyrate. The N-(2-methylphenyl) butyryl hydroxamate (6)
product derived from 2-nitrosotoluene has a calculated mo-
lecular weight of 179.0946, consistent with the found mo-
lecular ions at m/z = 180.1023 ([M+H]+) and m/z =
201.0836 ([M+Na]+). The N-phenyl butyryl hydroxamate (7)
product derived from nitrosobenzene has a calculated mo-
lecular weight of 165.0790, consistent with the found mo-
lecular ions at m/z = 166.0885 ([M+H]+) and m/z =
188.0720 ([M+Na]+). However, the conversion rate was not
determined due to decomposition of the nitroso compounds
in aqueous solution after prolonged incubation. Probably
due to the same instability of the nitroso compounds, no
product was found for other -ketoacids that might interact
with the enzyme too slowly to afford a detectable product.
3.5 Discussion
The MenD orthologue from E. coli has been shown to pos-
sess relaxed substrate specificities in using structurally di-
verse -ketoacids to generate the acyl anion intermediate
for addition to carbonyl electrophiles [22]. However, the
non-physiological reactions are slow, indicating that the
enzyme has achieved a high level of specificity for the na-
tive substrates. This is supported by the results presented
here. The observed lack of bsMenD activity towards cin-
namic acid and its derivatives is a testament for the high

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Chen MJ, et al. Sci China Chem March (2013) Vol.56 No.3
Table 3 The bsMenD-catalyzed CN bond ligation between nitroso compounds and -ketoacids a)
Donor Acceptor
Product
nd.b)
nd.
nd.
nd.
a) Reaction mixture contained 200 µM nitrosobenzene or 2-methyl nitrosobenzene, 2 mM 2-ketoacid, 100 µM ThDP, 268 nM MenD in 50 mM Tris buff-
er (pH 7.8) supplemented with 50 mM NaCl and 10 mM MgCl2 and was incubated at 37 C for 6 h; b) nd = not detected.
specificity of the enzyme for isochorismate. In addition, the
detected low reactivity of the non-physiological -ketoacid
substrates towards nitrosoarenes provides evidence for the
stringent structural requirements on the 2-oxo-glutarate sub-
strate. These experimental observations are fully consistent
with the crystallographic studies in which several substrate
recognition motifs have been identified in the enzyme active
site for each substrate [20, 21].
It was a total surprise that bsMenD catalyzes formation
of the N-alkyl hydroxamate from 4-nitrocinnamic acid and
2-oxo-glutarate. We propose a mechanism involving a re-
ductive C–N bond ligation reaction as shown in Figure 4. In
this mechanism, the enzyme-mediated decarboxylative ad-
dition of the 2-oxo-glutarate substrate with the ThDP co-
factor affords the acyl anion intermediate as usual, which
then reduces the oxidative nitro group in 4-nitrocinnamic
through transfer of two electrons. This electron transfer oxi-
dizes the acyl anion to acyl-thiazolium that subsequently
undergoes hydrolysis to recover the enzyme cofactor, while
the nitro compound is reduced to a nitroso intermediate. In
the next step, the recovered ThDP cofactor converts another
molecule of 2-oxo-glutarate to the acyl anion, which under-
goes nucleophilic attack on the nitroso group of the reduc-
tion product to eventually form the N-alkyl hydroxamate.
The proposed formation of the nitroso intermediate is
strongly supported by the much faster reaction of nitro-
soarenes with 2-oxo-glutarate (Figure 3) and 2-oxo-butyrate
to form similar N-alkyl hydroxamate products.
The proposed electron transfer could occur through bond
or through space. Considering that similar redox reaction
also occurs to bulky electron acceptors such as 6-dichloro-
phenolindophenol (DCPIP) (data not shown), through-space
electron transfer is more likely. However, the other possi-
bility cannot be ruled out. Suggested by the many inactive
nitro compounds listed in Table 2, this electron transfer is
likely dependent on the oxidative potential of the electron
acceptor, which has to be at least as high as nitrobenzene.
This possibility suggests that similar electron transfer reac-
tions should happen between the common acyl anion inter-
mediate of all ThDP-dependent enzymes and any oxidants
with an adequate oxidative potential, thereby opening up
new chemoenzymatic applications for the ThDP-enzymes in
redox chemistry. In addition, the observed bsMenD reduc-
tion of the nitro compounds also suggests that mitochondrial
ThDP-dependent enzymes may also be involved in reduc-
tion of organic nitrates such as nitroglycerin to activate this
class of important cardiovascular drugs in humans [28, 29].
4 Conclusions
Besides the aforementioned reduction reaction, the results
presented here also demonstrate for the first time the use of
nitroso compounds as electrophilic acceptor of the ThDP-

Chen MJ, et al. Sci China Chem March (2013) Vol.56 No.3
319
Figure 4 The proposed mechanism for the bsMenD-catalyzed reductive C–N bond ligation between 4-nitrocinnamic acid and 2-oxo-glutarate. SEPHCHC:
(1R, 2S, 5S, 6S)-2-succinyl-5-enolpyruvyl-6-hydroxyl-3-cyclohexene-1-carboxylate; Pi: phosphate group.
iaries: Diastereoselective Aldol reactions of a (1R, 2S)-ephedrine-
based 3,4,5,6-tetrahydro-2H-1,3,4-oxadiazin-2-one. Org Lett, 2002, 4:
3739–3742
derived acyl anion to form a new CN bond. It would be
interesting to see whether other ThDP-dependent enzymes
could also catalyze these reactions. At present, it is not
practical to use either reaction for chemoenzymatic synthe-
sis due to the detected limitations on the substrate structure.
To use these reactions, the substrate spectrum of the enzyme
has to be expanded. This could be achieved by modification
of the active site through site-directed mutagenesis and di-
rected evolution, similar to the many successful examples in
the literature [3033]. Thus, in combination with modern
bioengineering techniques, the new reactions found in this
work may eventually lead to new chemoenzymatic applica-
tions of the ThDP-dependent enzymes.
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