Assessing the stereoselectivity of: Serratia marcescens CECT 977 2,3-butanediol dehydrogenase

Article abstract of DOI:10.1039/c7cy00169j

α-Hydroxy ketones and vicinal diols constitute well-known building blocks in organic synthesis. Here we describe one enzyme that enables the enantioselective synthesis of both building blocks starting from diketones. The enzyme 2,3-butanediol dehydrogenase (BudC) from S. marcescens CECT 977 belongs to the NADH-dependent metal-independent short-chain dehydrogenases/reductases family (SDR) and catalyses the selective asymmetric reductions of prochiral α-diketones to the corresponding α-hydroxy ketones and diols. BudC is highly active towards structurally diverse diketones in combination with nicotinamide cofactor regeneration systems. Aliphatic diketones, cyclic diketones and alkyl phenyl diketones are well accepted, whereas their derivatives possessing two bulky groups are not converted. In the reverse reaction vicinal diols are preferred over other substrates with hydroxy/keto groups in non-vicinal positions.

Full text of DOI:10.1039/c7cy00169j

View Article Online
View Journal
Catalysis
Science &
Technology
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: R. Medici, H.
Stammes, S. Kwakernaak, L. Otten and U. Hanefeld, Catal. Sci. Technol., 2017, DOI:
10.1039/C7CY00169J.
This is an Accepted Manuscript, which has been through the
Volume 6 Number 1 7 January 2016 Pages 1–308
Royal Society of Chemistry peer review process and has been
accepted for publication.
Catalysis
Science &
Technology
Accepted Manuscripts are published online shortly after
www.rsc.org/catalysis
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
author guidelines.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
ISSN 2044-4753
standard Terms & Conditions and the ethical guidelines, outlined
in our author and reviewer resource centre, still apply. In no
event shall the Royal Society of Chemistry be held responsible
PAPER
Qingzhu Zhang et al.
Catalytic mechanism of C–F bond cleavage: insights from QM/MM
analysis of fluoroacetate dehalogenase
for any errors or omissions in this Accepted Manuscript or any
consequences arising from the use of any information it contains.
rsc.li/catalysis

Please do not adjust margins
Catalysis Science & Technology
Page 1 of 8
View Article Online
DOI: 10.1039/C7CY00169J
Journal Name
ARTICLE
Assessing the stereoselectivity of Serratia marcescens CECT 977
2,3-butanediol dehydrogenase†
Rosario Médici,a Hanna Stammes,a Stender Kwakernaak,a Linda G. Ottena and Ulf Hanefeld*a
Received 00th January 20xx,
Accepted 00th January 20xx
α-Hydroxy ketones and vicinal diols constitute well-known building blocks in organic synthesis. Here we describe one
enzyme that enables the enantioselective synthesis of both building blocks starting from diketones. The enzyme 2,3-
butanediol dehydrogenase (BudC) from S. marcescens CECT 977 belongs to the NADH-dependent metal-independent
short-chain dehydrogenases/reductases family (SDR) and catalyses the selective asymmetric reductions of prochiral α-
diketones to the corresponding α-hydroxy ketones and diols. BudC is highly active towards structurally diverse diketones
in combination with nicotinamide cofactor regeneration systems. Aliphatic diketones, cyclic diketones and alkyl phenyl
diketones are well accepted, whereas their derivatives possessing two bulky groups are not converted. In the reverse
reaction vicinal diols are preferred over other substrates with hydroxy/keto groups in non-vicinal positions.
DOI: 10.1039/x0xx00000x
www.rsc.org/
selective enzymes. In particular, the Bacillus stearothermophilus10
enzyme (2S,3S)-2,3-butanediol dehydrogenase (BDH, EC 1.1.1.76)
gave excellent enantioselectivities for a range of substrates towards
1. Introduction
Enantiomerically pure α-hydroxy ketones and vicinal diols
constitute a valuable group of molecules useful directly as fine
the S,S-diols. R,R-Diols could be obtained employing for instance
Saccharomyces cerevisiae BDH.11 This indicates that more enzymes
with similarly high specificities for other substrates and isomers
including the meso isomer should be available. The oxidation of the
meso isomer would be of particular interest. In principle, this
enzymatic transformation could afford enantio-enriched α-hydroxy
ketones with up to 100% yield rather than the 50% obtained
through a kinetic resolution approach.
chemicals or as building blocks for asymmetric synthesis of
bioactive compounds and agrochemicals.1,
2
Numerous
chemical and enzymatic strategies have been devised to
address this challenge.2 To date all chemical approaches3-5 and
most of the biochemical approaches6-8 only allow the
formation of one of the two chiral products: either the α-
hydroxy ketones or vicinal diols. A particular challenge is the
development of methodologies allowing for the targeted
selective synthesis of both α-hydroxy ketones and vicinal diols,
in particular with aliphatic side chains (Scheme 1).
O
R'
O
R"
R'
Acetoin reductases/2,3-butanediol dehydrogenases (EC 1.1.1.4
and 1.1.1.76) constitute a less explored group of enzymes. They
belong to the family of NADH-dependent metal-independent short-
chain dehydrogenases/reductases and are responsible for the
accumulation of 2,3-butanediols in high titers (>100 g L-1) during the
cultivations of species such as Klebsiella, Enterobacter, Serratia and
Bacillus.9 In general, 2,3-butanediol is not produced as a single
O
R"
O
OH
O
R"
R"
R"
R"
R'
R'
R'
O
isomer, but as
a mixture of meso-2,3-butanediol with (S,S)-
OH
butanediol (L-(+)-form) or (R,R)-2,3-butanediol (D-(-)-form).
Microorganisms that do not produce the meso diol, generate
optically enriched (R,R)-butanediol. The variations in optical purity
might be due to a single unselective enzyme or due to mixtures of
R'
OH
Scheme 1: Summary of enzymatic methods applied for the synthesis of
α-hydroxy ketones and vicinal diols, employing oxidoreductases,7, 12-14
lyases,15, 16 hydrolases17 and epoxide hydrolases.18 Double reduction of
α-diketones is achieved by a few oxidoreductases19-22 and enzymatic
cascade strategies involving two enzymatic steps.8, 23, 24
Here we describe the investigation of meso-2,3-butanediol
dehydrogenase (BudC) from Serratia marcescens CECT 977,
including its stereoselectivity and substrate range.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
Please do not adjust margins

Please do not adjust margins
Catalysis Science & Technology
Page 2 of 8
View Article Online
DOI: 10.1039/C7CY00169J
ARTICLE
Journal Name
Table 1: Kinetic data of purified BudC.a
2. Results and discussion
2.1 Cloning and heterologous expression of BudC
Vmax
Substrate
Km
(mM)
6.9±1
1.7±0.2 195.0
3.1±0.3 118.7
-
-
kcat
kcat/Km
(M-1s-1)
9.6 x103
1.1 x105
3.8 x104
-
(U mg-1)
(s-1)
66.6
meso-2,3-BDOb
diacetylc
140.7±5.2
412±11.0
250.8±3.1
-
-
In the present work, the budC gene encoding to a 2,3-BDH from S.
marcescencs CECT 977 (756 bp) was amplified from genomic DNA
and cloned into pET-28a in frame with the N-terminal His6-tag to
facilitate purification of the recombinant enzyme. The DNA
sequence exhibited a 91.8% identity with S. marcescens H30 strain25
and both proteins were identical at protein level. This means that
the production and purification described earlier25 is also valid for
the enzyme described here. We therefore concentrated on
substrate range and enantioselectivity.
(±)-acetoinc
(2S,3S)-2,3-BDOb
(2R,3R)-2,3-BDOb
-
-
-
a Reaction conditions: 0.16 mM/0.32 mM NADH/NAD+, 0.27 nM BudC, substrate
concentration 100 μM to 150 mM in potassium phosphate buffer (50 mM, pH
7.0) at 37ºC. Turnover numbers (kcat) are expressed per monomer of BudC.
b Oxidation reaction
c Reduction reaction
2.3 Bioconversions
2.2 Enzyme kinetics
When reduction reactions containing stoichiometric amounts of
substrate (diacetyl or acetoin), NADH (5 mM) and BudC (0.5 μM)
were performed, no product formation was observed in any case.
Variation of the NADH concentration revealed this to be due to
NADH inhibition. Although not previously reported for this enzyme,
NADH inhibition was observed at concentrations higher than 0.3
mM; 1 mM being sufficient for complete inhibition (ESI Figure 5).
The budC gene was successfully expressed in E. coli BL21 (DE3) star
cells and the enzyme was purified from E. coli cell-free extracts by
Ni-NTA affinity chromatography. Activity of crude extracts reached
14.4 U mg-1 using rac-acetoin (50 mM) as substrate at pH 7.0 in a
reductive assay (ESI Figure 1). SDS-PAGE gels revealed the
production of a protein of approximately 28 kDa in agreement with
the calculated molecular weight (28.4 kDa). Native gels confirmed
the tetrameric oligomerization state, yielding a band between 120-
140 KDa (ESI Figure 2) in line with the earlier results.25
Consistent with the previous results, nicotinamide cofactor
regeneration strategies are essential to achieve significant product
yields. Ethanol, 2-propanol, and acetone were tested as co-
substrates for substrate-coupled cofactor regeneration. However,
none of them was accepted by BudC (Table 2). An enzyme-coupled
approach was accomplished employing formate dehydrogenase and
glucose dehydrogenase (both as crude cell extracts) to recycle
NADH, and NADH oxidase (as crude cell extracts) to regenerate
NAD+. With these systems at hand, both the BudC catalysed
oxidation and reduction could be investigated.
BudC catalyses the reduction of rac-acetoin preferably at
weakly acidic pH 5.0 rather than neutral pH (975 U mg-1 vs 250 U
mg-1). However, kinetic parameters at pH 5.0 were difficult to
determine, yielding inconsistent data due to the low stability of the
enzyme at this pH (t1/2: 2.5 h, Figure 1).
100
80
Whereas BudC was S-selective for the reduction of diacetyl
yielding (S,S)-2,3-butanediol ((S,S)-2,3-BDO), rac-acetoin was
reduced to both meso-2,3-BDO and (S,S)-2,3-BDO. Here (R)-acetoin
was the preferred substrate and 15% (S)-acetoin remained
unconverted after 24 h. Thus, BudC showed a stereo-preference
consistent with meso-2,3-butanediol dehydrogenases with respect
to acetoin.
60
40
o
pH 5.0, 4
pH 7.0, 4
C
o
20
0
C
o
pH 7.0, 37
C
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Time (h)
In the oxidation reaction meso-2,3-BDO led to a complex
product mixture. Starting with the initial formation of (R)-acetoin
BudC catalysed its racemization within 24 h, concomitant with
oxidation and reduction reactions, yielded small amounts of (2S,3S)-
2,3-BDO as a side product.
Figure 1: Effect of the temperature and pH on BudC activity. Reaction
conditions: 0.16 mM NADH, 0.27 nM BudC, 50 mM rac-acetoin in potassium
phosphate buffer (50 mM, pH 7.0) or potassium acetate buffer (50 mM, pH 5.0)
at the corresponding temperatures.
In line with these observations, only kinetic parameters at pH
7.0 were determined (Table 1, ESI Figure 4, A-D). In the reductive
reaction diacetyl is the preferred substrate of BudC over acetoin,
while only meso-2,3-butanediol oxidation was catalysed by the
enzyme under the conditions assessed. Despite its protein
sequence identity with the enzyme from S. marcescens H30, the
specific activities obtained in our study with BudC were significant
higher than those reported earlier (up to 4.5-fold at pH 5.0)25 under
identical reaction conditions.
In agreement with the kinetic measurements (Table 1), (2S,3S)-
2,3-BDO was poorly converted to (S)-acetoin (<5% yield). No
measurable amounts of diacetyl were detected.
Overall, BudC is S-selective for the reduction reaction of diacetyl
to (2S,3S)-2,3-BDO but displays a preference for (R)-acetoin as a
substrate for the second reduction step, which is also reduced with
S-selectivity. In the reverse oxidation reaction meso-2,3-BDO was
converted under the tested conditions, while the other diols and
acetoin are poor substrates for the oxidation reaction (Scheme 2).
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Catalysis Science & Technology
Page 3 of 8
View Article Online
DOI: 10.1039/C7CY00169J
Journal Name
ARTICLE
Experiments were performed in triplicate and the data are presented as
means with standard deviations.
O
O
OH
b n.d.: not detected.
O
OH
OH
(S,S)-2,3-BDO
diacetyl
2.5 Analysis of BudC stereoselectivity
The reduction of compounds 1a-1f was further investigated
employing both NADH regeneration systems using formate
dehydrogenase or glucose dehydrogenase, depending the reaction
scale (Figure 2 and Scheme 3).
O
OH
+
chemical racemisation
O
OH
OH
OH
meso-2,3-BDO
rac-acetoin
Scheme 2: Specificity of BudC in the reduction and oxidation of 2,3-dioxygenated
butane.
2.4 Substrate scope
Figure 2: α-Diketones reduced by BudC
.
In order to broaden the applicability of BudC, we extended our
investigation to structurally diverse α-diketones and vic-diols.
Enzymatic activity was determined spectrophotometrically and the
results are presented in Table 2. In the reduction reaction 2,3-
diketones were good substrates of BudC, while the enzyme activity
decreases with the increase of alkyl chain length. Sterically more
demanding ketone moieties, such as 1,2-cyclohexanedione and 1-
phenyl-1,2-propanedione were also converted by BudC, while
bulky-bulky α-diketones, such as benzil, or α-hydroxy ketones (i.e.
benzoin) were not converted. Thus, BudC catalyses the reduction of
aliphatic symmetric and non-symmetric α-diketones, while its
activity is severely affected by the presence of bulky moieties next
to the carbonyl groups either due to steric hindrance (i.e. 1c, 1d and
1e) or conformational effects (1c and 1f).
Reaction products were isolated by column chromatography
and characterised by 1H and 13C NMR, optical rotation, and by
comparison with the products obtained under the same conditions
with a well characterised commercial enzyme ADH-380 (Evoxx,
Düsseldorf, Germany), which is known to yield the corresponding S-
stereoisomers. Moreover, ADH-380 catalysed reactions were used
as positive control.26 Slight differences in reaction yields and
stereoselectivities were observed depending on the regeneration
system employed (crude extracts of formate or glucose
dehydrogenase).
In the oxidation reaction non-vicinal diols such as 2,4-
pentanediol and 1,3-butanediol were no substrates of BudC. These
results are in agreement with those previously reported by Zhang et
al.25 indicating that vicinal diols (1,2-, 2,3- or 3,4) are required for
catalysis.
Scheme 3: Stereoselective reduction of 1,2-diketones using BudC from S.
marcescens CECT 977.
Table 2: Substrate scope of BudC from S. marcescens CECT 977. a
Compound
U mg-1
249.2±11.4
220.5±12.6
88.5±2.1
19.4.6±0.7
23.4±3.4
6.2±0.5
n.d.b
2,3-Pentanedione (1a) was reduced to the diol (2S,3S)-4a in
good yield and selectivity after 1h. After 24h the yield was improved
further but a slight loss of selectivity was observed (Table 3, Entry 1-
2, ESI Figures 7 and 11). The results are in line with those for
diacetyl (Scheme 2).
1a 2,3-pentanedione
1b 2,3-hexanedione
1c 3,4-hexanedione
1d 2,3-heptanedione
1e 1-phenyl-1,2-propanedione
1f 1,2-cyclohexanedione
R-benzoin
rac-benzoin
benzil
acetone
2,3-hexanedione (1b) was also rapidly reduced to the
corresponding α-hydroxy ketone, with (S)-3-hydroxy-2-hexanone
(3b) as the main reaction product (80% yield, 99% ee, Table 3, Entry
4), while 2,3-hexanediol was produced only in low yields. High
yields of 3b were also achieved at semi-preparative scale with a
minor loss of enantioselectivity (Table 3, Entry 5, ESI Figures 8 and
12). It should be noted that the less accessible 3-ketogroup was
selectively reduced and the reaction almost stopped completely
after a single reduction. The second reduction step was much
slower, allowing to selective production of the acyloin.
n.d.
n.d.
n.d.
<1.0
<1.0
n.d.
n.d.
2,4-pentanediol
1,3-butanediol
ethanol
2-propanol
Reaction conditions: 50 mM substrate, 0.16 mM/0.32 mM NADH/NAD+,
a
0.27 nM BudC in potassium phosphate buffer (50 mM, pH 7.0) at 37ºC.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
Catalysis Science & Technology
Page 4 of 8
View Article Online
DOI: 10.1039/C7CY00169J
ARTICLE
Journal Name
Table 3: Biocatalytic reduction of α-diketones with BudC and ADH-380 and NADH regeneration systems.a
Reg.
Syst.
Entry Substrate Conc. [mM]
Enzyme
2 [%]b ee [%]b 3 [%]
ee [%]
4 [%]
de [%]
ee [%]
Time (h)
1
2
1a
1a
1a
1b
1b
1c
1c
1c
1d
1d
1d
1d
1d
1e
10
10
10
10
50
10
50
10
10
10
50
10
BudC
BudC
FDH
FDH
FDH
FDH
GDH
FDH
GDH
FDH
FDH
GDH
GDH
FDH
-
-
-
-
-
71
90
92
<5
<5
79
<5
99
<5
<5
<5
65
95
(2S,3S)
(2S,3S)
99 (2S,3S)
ND
96c
94c
>99
-
1
-
-
-
24
24
24
48
24
1
3
ADH-380
BudC
-
-
-
-
4
<5
-
ND
80
90
-
99
5
BudC
-
96
ND
-
6
BudC
15
92
-
80 (S)
93 (S)
-
-
(2S,3S)
ND
92c
7
BudC
-
-
-
8
ADH-380
BudC
-
-
99 (2S,3S)
ND
99
-
24
24
24
48
24
9
31
46
20
29
91 (S)
97 (S)
89 (S)
99 (S)
57
40
68
-
99 (ND)
99 (ND)
99 (ND)
-
10
11
12
13
14
15
16
17
BudC
ND
BudC
ND
94c
99
99
99
ADH-380
(2S,3R)
99 (2S,3R)
10
10
10
10
10
10
10
ADH-380
BudC
GDH
FDH
FDH
FDH
FDH
FDH
FDH
<5
25
15
<5
-
ND
-
55
40
65
-
-
24
2
85 (S)
89 (S)
9
59 (1S,2S)
5 (1S,2S)
1e
1e
1f
1f
1f
BudC
85 (S)
83 (S)
30
36
66
66
81
24
24
2
ADH-380
BudC
ND
99 (S)
69 (1S,2S)
67 (1S,2S)
67 (1S,2S)
99 (1S,2S)
99
99
-
-
-
-
-
-
99
99
18
19
BudC
-
-
24
24
ADH-380
-
-
ND stands for not determined.
aReaction conditions: the corresponding diketone (1a-1f, 10 or 50 mM) was added to 50 mM potassium phosphate buffer pH 7.0 with the nicotinamide
cofactor (0.16 mM), BudC (2 U ml-1) and regeneration system (FDH or GDH, 2U mL-1).
bYields, ees and des were determined by chiral GC analysis (when possible).
cSmall peaks could not be assigned due to missing standards, therefore it was impossible to distinguish between ee and de.
A
modified reaction profile was observed with 2,3- (Table 3, Entry 14, ESI Figure 6). Longer incubation times increased
heptanedione (1d). A mixture of 2-hydroxy-3-heptanone (2d) and 3- the yield of 4e up to 30 %, but reduced the stereoselectivity (Table
hydroxy-2-heptanone (3d) was produced, the latter again as the 3, Entry 15). While the S-selectivity was maintained BudC
main reaction product (Table 3, Entries 9-10). As with 1b the less surprisingly cannot differentiate between a methyl and a phenyl-
accessible 3-ketogroup was thus reduced preferably. Despite group.
extensive efforts, the regio-isomers could not be separated by
1,2-cyclohexanedione was completely reduced to (S,S)-1,2-
cyclohexanediol (4f) in good yields (66 %) and with excellent ee
(99%) although poorer de (67%), as cis-1,2-cyclohexanediol was also
detected as a reaction product. It could not be distinguished
whether the low de was due to poor selectivity in the first or second
reduction step.
column chromatography; therefore the absolute stereochemistry of
3d could not be determined (ESI Figures 10 and 15-17). Based on
the other results it can tentatively be assigned as (S). (2S,3R)-2,3-
Heptanediol (4d) was obtained in low yields (<5 %). Neither longer
reaction times nor fresh pulses of the enzyme improved the yield of
the diol.
In contrast to other dehydrogenases (i.e., ADH-A from
20
Rhodococcus ruber or ADH-T from Thermoanaerobacter sp.),19,
2.6 Homology modelling of BudC and substrate docking
where the extension of α-diketone alkyl chain (n=1 to n=3)
increased the selectivity of the enzymes, a lower regio-selectivity
was observed in case of budC, indicating its preference for small
substrates.
An homology model of BudC was made based solely on the
structure of the meso-2,3-BDH from Klebsiella pneumoniae (KpBDH,
PDB 1GEG).27 This resulted in a model in which the positioning of
several active site residues is in line with another meso-BDH, which
seems important for the enantioselectivity of the enzyme.28 The
model shows the enzyme as a tetramer, which is in accordance with
the results from the native PAGE and other SDR family members
(see Electronic Supplementary Information Figures 18 and 19). The
important active site residues for SDRs (Asn-Ser-Tyr-Lys) are in the
exact same position as in other BDHs, the only difference is Asp111
in the place were normally an Asn is located. This residue plays an
essential role in keeping the conformation of the active site and
allowing a proton relay system,29 which is still there involving the
Asp residue.
The reduction of 3,4-hexanedione (1c) proceeded with the
same stereoselectivity as for diacetyl, (S)-acetoin and 1a yielding
(3S,4S)-3,4-hexanediol (4c) (79% yield, 92% stereoselectivity). When
higher substrate concentrations were employed and the reaction
time shortened (Table 3, Entries 7), (S)-4-hydroxy-3-hexanone (2c)
was produced with excellent yields (92%) and stereoselectivity (93%
ee). This supports the stereochemical assignment of 3d (ESI Figures
8 and 13-14).
1-Phenyl-1,2-propanedione reduction yielded (S)-2-hydroxy-1-
phenylpropan-1-one (3e) as the main product (55% yield, 89% ee),
(S)-1-hydroxy-1-phenylpropan-2-one (2e, 25% yield, 85% ee) and
(S,S)-1-phenyl-1,2-propanediol (4e) (9% yield, 99% ee, 59% de)
Docking different substrates and products did not result in good
binding poses. This is probably due to the small nature of the
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Catalysis Science & Technology
Page 5 of 8
View Article Online
DOI: 10.1039/C7CY00169J
Journal Name
ARTICLE
substrates. This was confirmed by the fact that docking these small
compounds into the crystal structure of the KpBDH also did not
result in proper binding modes. Furthermore, the loop just outside
the binding pocket is probably moving upon binding of the
substrate which cannot be mimicked by the docking in silico.
4. Experimental
4.1 Strains, plasmids and culture conditions
All standard recombinant DNA procedures were performed as
described by Sambrook et al.30 The host strains Escherichia coli
DH5α (Thermo Fisher Scientific, Breda, The Netherlands) and
Escherichia coli BL21 star (DE3) (Thermo Fisher Scientific) were used
in sub-cloning and expression experiments, respectively.
Normally the reduction of the proximal keto group results in the
S-enantiomer of the corresponding α-hydroxy ketone. The pro-R
NADH hydride transfer from NADH to the re-face of diketones yields
(S)-hydroxy ketones, indicating that the enzyme obeys canonical
Prelog addition (Figure 3). Small diketones are most probably fixed
at the correct distance to NADH with hydrogen bonds, similar to
KpBDH. Since the active site easily can accommodate larger
substrates, the larger and more aliphatic penta-, hexa- and
heptadione can bind into the active site in an alternative
orientation. In this way the 3-keto group may be positioned
properly towards the NADH, resulting in the respective 3S-
hydroxyketone. Reduced affinity of this alternative binding mode
can explain the decreased activity for the larger substrates.
Serratia marcescens CECT 977 was supplied by the Spanish Type
Culture Collection (CECT, University of Valencia, Spain) and as DNA
source for budC cloning. pGEMT-easy (Promega, Promega Benelux
B.V., Leiden, The Netherlands) was used as cloning vector and pET-
28a (Merck Millipore, Amsterdam, The Netherlands) as expression
vector.
Bacteria were routinely grown at 37 °C in liquid Luria-Bertani broth
medium (NaCl, 5 g L−1; tryptone, 10 g L−1; yeast extract, 5 g L−1)
containing 100 μg ml-1 ampicillin or 50 μg ml-1 kanamycin when
required.
3. Conclusions
4.2 Construction of pET-28a budC expression vector
The BudC gene was successfully cloned and recombinantly
expressed in E. coli. The enzyme is (S)-selective for all tested
substrates and has a broad substrate scope. Unlike other ADHs it
displays a better stereoselectivity for smaller substrates. Although
the enzyme is strictly S-specific for all small substrates, meso-2,3-
BDO is produced with excellent selectivity using Serratia
marcescens strains. The production of this compound can be
explained by the accumulation of R-acetoin in these strains, which
is a very good substrate for the S-specific BudC.
By varying reaction conditions, single or double reductions as
well as oxidations could be achieved in one step. This makes this
enzyme an outstanding biocatalyst for the synthesis of different
chiral compounds from prochiral diketones and diols.
S. marcescens genomic DNA was extracted following a standard
phenol/chloroform procedure as reported by Sambrook et al.,
(2001). A budC consensus sequence was generated based on the
alignment of homologous sequences from five S. marcescens strains
(S. marcescens SM39, accession number: AP013063; S. marcescens
MG1, accession number: JF519738; S. marcescens subsp.
marcescens Db11, accession number HG326223; S. marcescens
G12, accession number: KF547938; S. marcescens WW4, accession
number: CP003959) with budC gene sequence of S. marcescens H30
(accession number: JQ63959) reported by Zhang et al.,25 as
Figure 3: Homology model of BudC based on meso-2,3-butanediol dehydrogenase from K. pneumoniae (1GEG). The substrate diacetyl (blue sticks)
was modelled based on the co-crystalised beta-mercaptoethanol in 1GEG. Tyr155 and Ser138 (yellow) are both hydrogen bonded to the proximal
keto-group. The Tyr donates a hydride to the oxygen of the substrate, while the pro-R hydride from NADH (orange) transfers to the carbon, resulting
in the S-configuration of the hydroxyl group.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
Please do not adjust margins

Please do not adjust margins
Catalysis Science & Technology
Page 6 of 8
View Article Online
DOI: 10.1039/C7CY00169J
ARTICLE
Journal Name
template.
6.22 mM-1 cm-1. One unit of activity was defined as the amount of
enzyme catalysing the oxidation/reduction of μmol of
1
The budC gene was PCR amplified from S. marcescens CECT 977
nicotinamide cofactor per minute under standard conditions (pH
7.0, 37°C).
genomic
GGAATTCCATATGCGTTTTGACAATAAAGTCGTGGTTATC -3’ (NdeI site
in bold) and reverse primer
DNA
using
the
forward
5’-
5’- Specific activities were measured in potassium phosphate buffer
CGCTCGAGTTAGACGATCTTCGGTTGGCCGTCCGA-3’ (XhoI site in (50 mM, pH 7.0, 1 ml) in presence of 50 mM of substrate and 0.16
bold). The PCR was performed using Accuprime Pfx DNA mM NADH or 0.32 mM NAD+. DMSO (5% (v/v)) was added as a co-
polymerase (Thermo Fisher Scientific) with the following conditions: solvent to improve the solubility of benzil and benzoin. Reactions
0.1 µg of chromosomal DNA, 0.3 µM of each primer, 5 µl of 10x PFx were started by addition of the enzyme solution and measured for
reaction mix and 2.5 units of Pfx DNA polymerase in a final volume 3 min. Activities of the CFE was determined using E. coli BL21 (DE3)
of 50 µl. A hot start of 2 minutes at 95˚C was followed by 30 cycles star pET28(a)-buC or E. coli BL21 (DE3) star pET28(a) as negative
of denaturation (15 s at 95 ˚C), annealing (30 s at 56 ˚C), and control.
extension (1 min at 68 ˚C). The PCR product was first purified using
Kinetics parameters were investigated using 0.27 nM enzyme in
presence of varying substrate (0.01-200 mM) or nicotinamide
cofactor concentrations (12.5 μM to 5 mM) at 37 °C. Data were
a Gel purification kit (Qiagen, Venlo, The Netherlands), followed by
an A-tailing procedure with Taq DNA polymerase (Qiagen).
BudC gene was ligated into pGEM®T-Easy vector system (Promega, fitted to the Michaelis-Menten equation (non-linear regression,
Leiden, The Netherlands), and the resulting plasmids were SigmaPlot 8.0) to estimates of Km and kcat
.
produced in E. coli DH5α. The resulting constructs were confirmed
4.5 Standard reaction conditions
by DNA sequencing, restricted with NdeI and XhoI, and the coding
gene was ligated into the corresponding sites of pET-28a vector
(Merck Millipore, Amsterdam, The Netherlands), introducing a
(His)6-tag at the N-terminus of budC gene product.
The standard reduction mixture consisted of 10 mM diketones, 0.16
mM of NADH, 2 U ml-1 of BudC in potassium phosphate buffer (50
mM, pH 7.0) and NADH-regeneration system containing 100 mM
ammonium formate and
2 U
ml-1 formate dehydrogenase
Recombinant expression of budC was performed using E. coli BL21
star (DE3) cells transformed with pET-28a-budC in autoinduction
media (ZYM-5052)31 supplemented with 100 μg ml-1 of kanamycin
at 22°C and 150 rpm during 24 h. Cells were harvested by
centrifugation at 12,500 x g for 15 minutes at 4°C, washed twice
with potassium phosphate buffer (50 mM, pH 7.0), re-centrifuged
stored at -80°C until further use.
(Prozomix, Northumberland, UK). Oxidation reactions comprised:
10 mM diols, 0.32 mM NAD+, 2 U ml-1 of BudC in potassium
phosphate buffer (50 mM, pH 7.0) and NAD+-regeneration system
containing 4 U ml-1 of NADH-oxidase (Prozomix).
Reactions were incubated at 37 ºC and 600 rpm during 24 h.
Aliquots (1 ml) were taken at different reaction times, centrifuged
at 13000 rpm for 2 minutes and supernatants were first saturated
with a NaCl saturated solution (0.1 ml) followed by extraction with
4.3 BudC purification
Cell-free extracts (CFE) were obtained incubating first the cells in ethyl acetate (containing isoamyl alcohol as internal standard) (0.5
binding buffer (sodium phosphate 50 mM, 300 mM NaCl, 20 mM mL x 2). The combined organic layer was dried over Na2SO4 and
imidazole, pH 8.0) containing egg white lysozyme (1 mg ml-1) and analysed with chiral GC with respect to yield, chemoselectivity and
DNAse I (0.1 mg ml-1) for 1 h at 4°C, followed by 2 cycles of cell ee (see Electronic Supplementary Information).
disruption (French Press, 1.5 kBar) and finally centrifuged at 12,500
Aliphatic α-hydroxy ketones and diol standards were obtained
carrying out the reduction reactions at 50 ml scale. The reaction
mixture contained 50 mM substrate (200 mg 2,3-pentanedione, 285
mg 2,3-hexanedione, 285 mg 3,4-hexanedione, 320 mg 2,3-
heptanedione), 7.1 mg NADH, 1.5 g glucose-1-monohydrate, 0.75 g
calcium carbonate, 2 mg glucose dehydrogenase and 2 mg ADH-380
(Evoxx) or 1.4 mg of BudC in potassium phosphate buffer (50 mM,
x g and 4°C for 45 min. CFE was filtered through 0.45 µm filter and
applied onto a 5 ml Ni-NTA HisTrap HP column (GE Healthcare Life
Sciences, Eindhoven, The Netherlands) previously equilibrated with
binding buffer. An imidazole gradient was then applied (20 mM-500
mM imidazole, 3 ml min-1, 60 minutes) and BudC was eluted with
about 350 mM of imidazole.
Enzyme fractions were analysed by SDS-PAGE (12% Bis-Tris gels, pH 7.0). Reaction vessels were incubated at 37 °C and 100 rpm
Bio-Rad, Munich, Germany). Fractions containing BudC were during 24 or 48 h. α-Hydroxy ketones and diols were purified by
combined, concentrated by centrifugation using an Amicon filter column chromatography as detailed in ESI (sections 3 and 4) ,
1
(10 kDa, Merck Millipore) and applied onto a PD-10 desalting characterised by their optical rotation, H and 13C NMR spectra and
column (Thermo Fischer Scientific) previously equilibrated with 50 compared with authentic standards or literature data when
mM potassium phosphate, pH 7.0. Enzyme aliquots were frozen in available (see ESI Figures 6 to 11).
liquid nitrogen and stored at -80°C. Protein concentration was
4.6 Homology modelling of BudC and docking of substrates
determined by the bicinchoninic acid assay (Interchim Uptima BC
assay, Interchim, Montluçon France) using bovine serum albumin
(Bio-Rad, Veenendaal, The Netherlands) as standard.
An homology model of BudC was build using the standard
homology parameters of Yasara.32 Only meso-2,3-BDH (1GEG) was
used as a template in order to preserve the position of catalytically
important side chains.
4.4 Enzyme kinetics
Enzymatic activity was determined spectrophotometrically by
monitoring NADH consumption (oxidation) or formation (reduction)
at 340 nm and 37 °C using an extinction coefficient of
Docking was done in the Yasara program with 100 docking runs
using VINA and a minimum ligand RMSD of 1.5 Å. All residues were
6 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Catalysis Science & Technology
Page 7 of 8
View Article Online
DOI: 10.1039/C7CY00169J
Journal Name
ARTICLE
fixed except the important residues lining the active site, namely 17.
Ser138, Val139, Trp146, Tyr151, Lys155, Trp192 and the
nicotinamide part of NADH. All docked compounds were visually
inspected to determine the most probable catalytically active 18.
position. This binding mode was minimised with the NOVA force
A. Petrenz, P. D. d. María, A. Ramanathan, U. Hanefeld, M.
B. Ansorge-Schumacher and S. Kara, J. Mol. Catal. B:
Enzym., 2015, 114, 42-49.
M. Kotik, A. Archelas and R. Wohlgemuth, Curr. Org.
Chem., 2012, 16, 451-482.
field to improve binding interactions with the protein.
19.
K. Edegger, W. Stampfer, B. Seisser, K. Faber, S. F. Mayer,
R. Oehrlein, A. Hafner and W. Kroutil, Eur. J. Org. Chem.,
2006, 2006, 1904-1909.
20.
21.
22.
M. Kurina-Sanz, F. R. Bisogno, I. Lavandera, A. A. Orden
and V. Gotor, Adv. Synth. Catal., 2009, 351, 1842-1848.
P. Besse, J. Bolte, A. Fauve and H. Veschambre, Bioorg.
Chem., 1993, 21, 342-353.
L. N. Monsalve, P. Cerrutti, M. A. Galvagno and A.
Baldessari, Biocatal. Biotransform., 2010, 28, 137-143.
Acknowledgements
This study was financially supported by The Netherlands
Organization for Scientific Research (NWO) under the framework of
Technology Area TA-Biomass. We thank Martina Pohl and Dörte
Rother
(Forschungszentrum
Jülich
GmBH,
IBG-1:
Biotechnology, Jülich, Germany) for providing 1-phenyl-1,2- 23.
J. Zhang, S. Wu, J. Wu and Z. Li, ACS Catal., 2015, 5, 51-58.
24.
25.
A. Jakoblinnert and D. Rother, Green Chem., 2014, 16,
3472-3482.
L. Zhang, Q. Xu, S. Zhan, Y. Li, H. Lin, S. Sun, L. Sha, K. Hu,
X. Guan and Y. Shen, Appl. Microbiol. Biotechnol., 2014,
98, 1175-1184.
propanediol and 2-hydroxy-1-phenylpropan-1-one isomers and
the enzymes for the synthesis of 1-hydroxy-1-phenylpropan-2-
ones and for revising the manuscript.
26.
Evocatal
GmbH,
References
http://www.biocatal.com/download.php?dl=adh380,
(accessed 16-12-2016, 2016).
M. Otagiri, S. Ui, Y. Takusagawa, M. Ohkuma, T. Kudo and
M. Kusunoki, J. Biochem., 2001, 129, 205-208.
M. Otagiri, S. Ui, Y. Takusagawa, T. Ohtsuki, G. Kurisu and
M. Kusunoki, FEBS Lett., 2010, 584, 219-223.
C. Filling, K. D. Berndt, J. Benach, S. Knapp, T. Prozorovski,
E. Nordling, R. Ladenstein, H. Jörnvall and U. Oppermann,
J. Biol. Chem., 2002, 277, 25677-25684.
J. Sambrook, E. F. Fritsch and T. Maniatis, Molecular
cloning:
Laboratory Press, New York, Third edn., 2001.
F. W. Studier, Protein Expression Purif., 2005, 41, 207-234.
E. Krieger and G. Vriend, Bioinformatics, 2014, 30, 2981-
2982.
1.
2.
3.
4.
5.
C. Palomo, M. Oiarbide and J. M. Garcia, Chem. Soc. Rev.,
2012, 41, 4150-4164.
27.
28.
29.
P. Hoyos, J. V. Sinisterra, F. Molinari, A. R. Alcantara and P.
Dominguez de Maria, Acc. Chem. Res., 2010, 43, 288-299.
C. R. Unelius, B. Bohman, M. G. Lorenzo, A. Troger, S.
Franke and W. Francke, Org. Lett., 2010, 12, 5601-5603.
B. Plietker, Tetrahedron: Asymmetry, 2005, 16, 3453-
3459.
S. E. Schaus, B. D. Brandes, J. F. Larrow, M. Tokunaga, K. B.
Hansen, A. E. Gould, M. E. Furrow and E. N. Jacobsen, J.
Am. Chem. Soc., 2002, 124, 1307-1315.
30.
a laboratory manual, Cold Spring Harbor
31.
32.
6.
7.
P. P. Giovannini, O. Bortolini and A. Massi, Eur. J. Org.
Chem., 2016, 2016, 4441-4459.
J. Kulig, R. C. Simon, C. A. Rose, S. M. Husain, M. Häckh, S.
Lüdeke, K. Zeitler, W. Kroutil, M. Pohl and D. Rother,
Catal. Sci. Technol., 2012, 2, 1580.
8.
S. T. Kihumbu D, Hummel W, Liese A., Tetrahedron:
Asymmetry, 2002, 13, 1069-1072.
9.
E. Celinska and W. Grajek, Biotechnol. Adv., 2009, 27, 715-
725.
10.
O. Bortolini, G. Fantin, M. Fogagnolo, P. P. Giovannini, A.
Guerrini and A. Medici, J. Org. Chem., 1997, 62, 1854-
1856.
11.
E. Calam, E. Gonzalez-Roca, M. R. Fernandez, S. Dequin, X.
Pares, A. Virgili and J. A. Biosca, Appl. Environ. Microbiol.,
2016, 82, 1706-1721.
12.
13.
14.
15.
16.
E. Calam, S. Porte, M. R. Fernandez, J. Farres, X. Pares and
J. A. Biosca, Chem. -Biol. Interact., 2013, 202, 195-203.
C. Loderer and M. B. Ansorge-Schumacher, RSC Adv.,
2015,
C. E. Paul, I. Lavandera, V. Gotor-Fernández, W. Kroutil
and V. Gotor, ChemCatChem, 2013, , 3875-3881.
5, 38271-38276.
5
M. Muller, D. Gocke and M. Pohl, FEBS J., 2009, 276
,
2894-2904.
P. Domínguez de María, M. Pohl, D. Gocke, H. Gröger, H.
Trauthwein, T. Stillger, L. Walter and M. Müller, Eur. J.
Org. Chem., 2007, 2007, 2940-2944.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
Please do not adjust margins

Catalysis Science & Technology
Page 8 of 8
View Article Online
DOI: 10.1039/C7CY00169J
The meso-2,3-butanediol dehydrogenase from Serratia marcescens CECT 977 can produce several
optically active hydroxy ketones from both the alcohol or ketone precursor.