Elucidation of the enantioselective cyclohexane-1,2-dione hydrolase catalyzed formation of (S)-acetoin

Article abstract of DOI:10.1002/cctc.201300904

Thiamine diphosphate (ThDP) dependent enzymes catalyze the formation of acetoin (3-hydroxybutan-2-one) through one of three different pathways: homocoupling of pyruvate, homocoupling of acetaldehyde, or cross-coupling of acetaldehyde (as acceptor) and pyruvate (as donor). The enantioselectivity of the resulting acetoin is highly dependent on the particular enzyme. We established that ThDP-dependent cyclohexane-1,2-dione hydrolase (CDH) is able to form (S)-acetoin with particularly high enantioselectivity (up to 95 % ee) by all three pathways. Mechanistic studies utilizing ¹³C-labeled substrates revealed an unprecedented non-acetolactate pathway for the homocoupling of pyruvate, which explains the high enantioselectivity in the CDH-catalyzed formation of (S)-acetoin. Differentiating hydrolases: Investigating thiamine diphosphate dependent cyclohexane-1,2-dione hydrolase (CDH) catalyzed homocoupling of ¹³C-labeled [1,2]-¹³C-pyruvate to (S)-[2,3]-¹³C-acetoin reveals a non-acetolactate pathway, which explains the high enantioselectivity of this reaction (up to 93 % ee). CDH also catalyzes the formation of (S)-acetoin by the cross-coupling of pyruvate and acetaldehyde and the homocoupling of acetaldehyde.

Full text of DOI:10.1002/cctc.201300904

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DOI: 10.1002/cctc.201300904
Elucidation of the Enantioselective Cyclohexane-1,2-dione
Hydrolase Catalyzed Formation of (S)-Acetoin
Sabrina Loschonsky, Simon Waltzer, Volker Brecht, and Michael Mꢀller*[a]
Thiamine diphosphate (ThDP) dependent enzymes catalyze the
formation of acetoin (3-hydroxybutan-2-one) through one of
three different pathways: homocoupling of pyruvate, homo-
coupling of acetaldehyde, or cross-coupling of acetaldehyde
(as acceptor) and pyruvate (as donor). The enantioselectivity of
the resulting acetoin is highly dependent on the particular
enzyme. We established that ThDP-dependent cyclohexane-
1,2-dione hydrolase (CDH) is able to form (S)-acetoin with par-
ticularly high enantioselectivity (up to 95%ee) by all three
pathways. Mechanistic studies utilizing 13C-labeled substrates
revealed an unprecedented non-acetolactate pathway for the
homocoupling of pyruvate, which explains the high enantiose-
lectivity in the CDH-catalyzed formation of (S)-acetoin.
(R)-1 (46–53%ee),[7] whereas PDC from Zymomonas mobilis
(ZmPDC) forms (S)-1 (23–29%ee).[7,8] A single mutation
(ZmPDC-E473Q) results in inversion of the stereoselectivity
[(R)-1, 33%ee].[8] Whereas YerE from Yersinia pseudotubercu-
losis yields almost racemic acetoin (4%ee),[9] PigD from Ser-
ratia marcescens provides the (S) enantiomer (70%ee).[10]
2) Homocoupling of acetaldehyde is known to be catalyzed
by PDC from several organisms, that is, Acetobacter pasteur-
ianus [(S)-1, 28%ee][11] and its variant ApPDC-E469G [(S)-1,
85%ee],[11] Zymobacter palmae [(S)-1, 58%ee],[12] Z. mobilis
[(S)-1, 25%ee],[7] and yeast [(R)-1, 44%ee].[7] Moreover, ben-
zoylformate decarboxylase (BFD) from Pseudomonas putida
[(R)-1, 17%ee][11] and its variant PpBFD-H281A [(R)-1,
25%ee],[11] benzaldehyde lyase (BAL) from Rhodopseudomo-
nas palustris [(S)-1, 60%ee],[12] and branched-chain keto
acid decarboxylase (KdcA) from Lactobacillus lactis [in
buffer: (R)-1, 20%ee; in the presence of 10–30% acetone:
(S)-1, 21%ee][11] catalyze the same reaction.
Acetoin (1) is produced by a variety of (micro)organisms that
metabolize glucose through the Embden–Meyerhof–Parnas
pathway, the most important form of glycolysis for the trans-
formation of d-(+)-glucose into pyruvate (2).[1] The most
common biosynthetic pathway leading to acetoin involves the
formation of acetolactate (3) by the homocoupling of pyruvate
catalyzed by acetolactate synthase.[2] Acetolactate is subse-
quently transformed into acetoin by acetolactate decarboxy-
lase. Alternatively, acetoin can also accumulate as a byproduct
of pyruvate decarboxylase[3] and pyruvate oxidase[4] catalysis.[2a]
Acetoin is found in many dietary products, such as butter,
apples, and yogurt, amongst others. Large-scale production of
acetoin has been achieved by organic synthesis, microbial fer-
mentation, and enzymatic transformation.[5]
3) PDC from yeast catalyzes the cross-coupling of acetalde-
hyde and pyruvate to give (R)-1 with 46–50%ee.[7] After
single-point mutations, the enantioselectivity can be in-
creased up to 94%ee [(R)-1].[13] ZmPDC yields (S)-1 with 28–
29%ee.[7] A variant of pyruvate dehydrogenase complex E1
subunit (PDHc-E1) from E. coli gives (S)-1 with up to
70%ee.[13]
Cyclohexane-1,2-dione hydrolase (CDH), isolated from Azoar-
cus sp. strain 22 Lin,[14,15] is one of the many ThDP-dependent
enzymes that catalyze asymmetric CꢀC bond formation from
pyruvate (as donor) and an aldehyde (as acceptor).[16] In this
work, we show that CDH is able to form acetoin with surpris-
ingly high enantioselectivity by all three aforementioned
modes. The results of our mechanistic studies with labeled and
unlabeled substrates clearly show that enzymatic formation of
highly enantioenriched acetoin from two molecules of pyru-
vate occurs without the release of acetaldehyde or acetolac-
tate. This unexpected observation is also of importance for
other ThDP-dependent asymmetric CꢀC bond-formation
reactions.
Thiamine diphosphate (ThDP) dependent enzymes catalyze
a broad range of reactions,[6] including (asymmetric) CꢀC bond
formation from an aldehyde and an a-keto carboxylic acid to
yield an a-hydroxy ketone. Acetoin (1) is such an a-hydroxy
ketone and can emerge from ThDP-dependent enzyme cataly-
sis through homocoupling of pyruvate, homocoupling of acet-
aldehyde, or the combination of acetaldehyde (as acceptor)
and pyruvate (as donor):
1) Pyruvate decarboxylase (PDC) from yeast (Saccharomyces
cerevisiae) catalyzes the homocoupling of pyruvate to yield
If only pyruvate is present, CDH catalyzes the formation of
(S)-acetoin with 87–90%ee after 24 h at 308C.[17] Upon per-
forming the reaction at 168C, 93%ee was obtained (see the
Supporting Information).[18] The homocoupling of 13C-labeled
[1,2]-13C-pyruvate (2a; Scheme 1, top) was followed by
13C NMR spectroscopy measurements every 30 min for 19.5 h
[2-(N-morpholino)ethanesulfonic acid (MES) buffer, 10% D2O,
v/v; see the Supporting Information]. [1,2]-13C-Pyruvate (2a)
showed two doublets (1J=62.2 Hz) at d=170.2 and
205.0 ppm, in addition to minor amounts of its hydrate form
[a] S. Loschonsky, S. Waltzer, V. Brecht, Prof. Dr. M. Mꢀller
Institute of Pharmaceutical Sciences
University of Freiburg
Albertstraße 25, 79104 Freiburg (Germany)
Fax: (+49)761-203-6351
E-mail: michael.mueller@pharmazie.uni-freiburg.de
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cctc.201300904. It contains the experimental
details for the reactions in Schemes 1–4.
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of acetaldehyde (4a) as the ac-
ceptor. The latter two species
must arise by initial decarboxyla-
tion of pyruvate (2a!4a·ThDP)
and
subsequent
protolysis
(!4a). This pathway might
serve as an explanation for the
remarkably high ee of (S)-ace-
toin, as the alternative pathway
via acetolactate (3) must by ne-
cessity incorporate (nonenzymat-
ic) decarboxylation of 3 to yield
the achiral enediol tautomer of
acetoin, which would tautomer-
ize to (racemic) acetoin in a final
step. To prove this, we investi-
gated the putative CDH-cata-
lyzed formation of acetoin from
Scheme 1. CDH-catalyzed formation of [2,3]-13C-acetoin (1a) from [1,2]-13C-pyruvate (2a). Conditions: [1,2]-13C-
pyruvate (25 mm), CDH (0.7 mgmLꢀ1), buffer A (50 mm MES, 1.5 mL; 1 mm MgSO4; 0.5 mm ThDP; 10% D2O;
pH 6.5], 308C. Conversion after 5 h is shown. Enantiomeric excess was determined by GC analysis on a chiral sta-
tionary phase (87–90% ee after 24 h by using unlabeled pyruvate).
a
mixture of [2]-13C-pyruvate
(2b) and acetaldehyde (4). As-
suming that both pyruvate and
acetaldehyde can act as a donor
and, moreover, that both acetal-
dehyde (4) and [1]-13C-acetalde-
hyde [(4a), formed by decarbox-
ylation of [2]-13C-pyruvate (2b)]
can act as acceptors, four isoto-
pologues, that is, 1a–d, are pos-
sible (Scheme 2, top).
However, we observed only
isotopologues 1a and 1b in
a ratio of approximately 10:90.
This was ascertained by integra-
tion of the doublet of [2,3]-13C-
acetoin (1a) at d=215.4 ppm
(1J2,3 =41.2 Hz) and the superim-
posed, yet baseline-separated,
singlet of [2]-13C-acetoin (1b) at
d=215.4 ppm. The overall con-
version after 18.5 h was 74%
based on [2]-13C-pyruvate (2b,
Scheme 2). (S)-Acetoin was ob-
tained with 89%ee (as deter-
mined after 24 h at 308C by
Scheme 2. CDH-catalyzed formation of isotopologues 1a–d from a mixture of [2]-13C-pyruvate (2b) and acetalde-
hyde (4). Conditions: [2]-13C-pyruvate (25 mm), acetaldehyde (15 mm), CDH (0.7 mgmLꢀ1), buffer A (1.5 mL), 308C.
13C NMR spectrum was recorded after 18.5 h (10% D2O, v/v). Enantiomeric excess was determined by GC analysis
on a chiral stationary phase (89% ee after 24 h by using unlabeled pyruvate and acetaldehyde).
with doublets (1J=63.1 Hz) at d=93.9 and 178.5 ppm. Acetoin
was formed as its [2,3]-13C-isotopomer 1a, which displays two
doublets (1J=41.2 Hz) at d=73.1 and 215.4 ppm. After 19.5 h,
90% conversion of [1,2]-13C-pyruvate (2a) into [2,3]-13C-acetoin
(1a) was observed [calculated from the 13C NMR spectrum by
integration of the 13C(=O) signals of 1a and 2a]. As acetolac-
tate is commonly referred to as a key intermediate in the
enzyme-catalyzed formation of acetoin from pyruvate, we
were surprised that [1,2,3]-13C-acetolactate (3a) could not be
detected in the 13C NMR spectra [i.e., at d=212 (C=O), 177
(CO2H), and 83 ppm (CꢀOH)].
using unlabeled pyruvate and acetaldehyde). If the reaction
was performed at 168C, 92%ee was obtained. From this out-
come, we conclude that pyruvate is a superior donor than
acetaldehyde, whereas acetaldehyde is a superior acceptor
than pyruvate.
Subsequently, we investigated the CDH-catalyzed formation
of [1,2,3,4]-13C-acetoin (1e) from [1,2]-13C-acetaldehyde (4b) as
the sole C2 source (Scheme 3). There was approximately 38%
conversion into product 1e after 21 h at 308C, as judged from
the 13C NMR spectrum (integration of the 13CH3 signals of 1e,
4b, and 4b-hydrate; see the Supporting Information). (S)-Ace-
toin was obtained in 91% ee by using unlabeled acetaldehyde
after 24 h at 308C. If the reaction was performed at 168C,
Accordingly, we hypothesized that acetoin (1a) was formed
by carboligation of activated 4a·ThDP with a second molecule
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Scheme 3. CDH-catalyzed formation of [1,2,3,4]-13C-acetoin (1e) from [1,2]-13C-acetaldehyde (4b). Conditions: [1,2]-13C-acetaldehyde (23 mm), CDH
(0.7 mgmLꢀ1), buffer A (1.5 mL), 308C. 13C NMR spectrum was recorded after 20.5 h (10% D2O, v/v). Enantiomeric excess was determined by GC analysis on
a chiral stationary phase (91% ee after 24 h by using unlabeled acetaldehyde).
that the non-acetolactate path-
way for the homocoupling of
pyruvate suggested in Scheme 1
might indeed be viable. Howev-
er, it could not be ruled out that
CDH does form acetolactate as
an intermediate and that the
latter is converted into acetoin
faster than the timescale of the
13C NMR experiment. We there-
fore designed a competition ex-
periment by utilizing ThDP-de-
pendent YerE[9] (Scheme 4).
Recently, it was shown that
YerE transforms pyruvate into
(S)-acetolactate, which is fol-
lowed by slow (and presumably
nonenzymatic) decarboxylation
Scheme 4. Formation of [2,3]-13C-acetolactate (3b) from [2]-13C-pyruvate (2b) catalyzed by YerE and subsequent
addition of CDH. Conditions: [2]-13C-pyruvate (25 mm), YerE (1 mgmLꢀ1), buffer A (1.5 mL), 308C. After 2 h, an ali-
quot (250 mL) of the assay was removed and replaced by the same volume of CDH in buffer A. The final concen-
trations of the enzymes were 1.3 (CDH) and 0.83 mgmLꢀ1 (YerE). Enantiomeric excess was determined by GC
analysis on a chiral stationary phase (66% ee after 7 h, 42% ee after 26 h).
to yield almost-racemic acetoin
[<5%ee in potassium phos-
phate (KPi) buffer, pH 8.0].[9]
Upon following the YerE-cata-
lyzed homocoupling of [2]-13C-
pyruvate (2b) in aqueous KPi
95%ee was obtained. The identity of isotopologue 1e was un-
buffer by 13C NMR spectroscopy measurements every 30 min,
we detected the formation of [2,3]-13C-acetolactate (3b) after
1 h and the onset of [2,3]-13C-acetoin (1a) formation after 3 h
(9%ee after 20 h, see the Supporting Information). Because
CDH shows no activity in KPi buffer, whereas YerE is active in
MES buffer, the latter buffer was used for the competition ex-
periments. There was approximately 47% conversion of 2b
into [2,3]-13C-acetolactate (3b) with YerE after 1.5 h (Scheme 4,
ambiguously proven from the corresponding 13C NMR coupling
1
1
of each nucleus: d=18.3 (d, J4,3 =36.1 Hz; C-4), 24.9 (dd, J1,2
=
=
40.4 Hz, 2J1,3 =13.8 Hz; C-1), 73.1 (ddd, 1J3,2 =41.2 Hz, J3,4
1
2
1
1
36.1 Hz, J3,1 =13.9 Hz; C-3), 215.4 ppm (dd, J2,1 ꢁ J2,3 ꢁ40.7 Hz;
C-2).
Hence, acetaldehyde can act both as the acceptor and the
donor in the CDH-catalyzed formation of acetoin. This implies
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In summary, ThDP-dependent CDH catalyzes the enantiose-
lective formation of (S)-acetoin by homocoupling of pyruvate
[87–90 (308C), 93% ee (168C)], homocoupling of acetaldehyde
[91 (308C), 95%ee (168C)], and cross-coupling of pyruvate and
acetaldehyde [89 (308C), 92%ee (168C)]. Utilizing 13C-labeled
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formation of (S)-acetoin. Nevertheless, acetolactate is
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acetaldehyde (route 3) and can, in principle, be decarboxylated
through a nonenzymatic pathway to yield (racemic) acetoin.
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formation of acetoin by either pathway explains the high
enantioselectivity in the formation of (S)-acetoin in these cases.
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Acknowledgements
This work was supported through the German Research Founda-
tion (DFG) (FOR 1296).
Keywords: asymmetric synthesis · biocatalysis · carboligation ·
cross-coupling · thiamine-dependent enzymes
Received: October 22, 2013
Published online on January 28, 2014
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