Catalytic scope of the thiamine-dependent multifunctional enzyme cyclohexane-1,2-dione hydrolase

Article abstract of DOI:10.1002/cbic.201300673

The thiamine diphosphate (ThDP)-dependent enzyme cyclohexane-1,2-dione hydrolase (CDH) was expressed in Escherichia coli and purified by affinity chromatography (Ni-NTA). Recombinant CDH showed the same Ci￡C bond-cleavage and Ci￡C bond-formation activitie

Full text of DOI:10.1002/cbic.201300673

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DOI: 10.1002/cbic.201300673
Catalytic Scope of the Thiamine-Dependent
Multifunctional Enzyme Cyclohexane-1,2-dione Hydrolase
Sabrina Loschonsky,[a] Simon Waltzer,[a] Sonja Fraas,[b] Tobias Wacker,[c]
Susana L. A. Andrade,[c] Peter M. H. Kroneck,[b] and Michael Mꢀller*[a]
The thiamine diphosphate (ThDP)-dependent enzyme cyclo-
hexane-1,2-dione hydrolase (CDH) was expressed in Escherichia
coli and purified by affinity chromatography (Ni-NTA). Recombi-
nant CDH showed the same CꢀC bond-cleavage and CꢀC
bond-formation activities as the native enzyme. Furthermore,
we have shown that CDH catalyzes the asymmetric cross-ben-
zoin reaction of aromatic aldehydes and (decarboxylated) pyru-
vate (up to quantitative conversion, 92–99% ee). CDH accepts
also hydroxybenzaldehydes and nitrobenzaldehydes; these
previously have not (or only in rare cases) been known as sub-
strates of other ThDP-dependent enzymes. On a semiprepara-
Scheme 1. ThDP-dependent cyclohexane-1,2-dione hydrolase (CDH) catalyz-
es A) CꢀC bond-cleavage and B) asymmetric CꢀC bond-formation.
tive scale, sterically demanding 4-(tert-butyl)benzaldehyde and
2-naphthaldehyde were transformed into the corresponding
2-hydroxy ketone products in high yields. Additionally, certain
benzaldehydes with electron withdrawing substituents were
identified as potential inhibitors of the ligase activity of CDH.
bacterium Azoarcus sp. strain 22Lin[7] when cultivated on cyclo-
hexane-1,2-diol as the sole carbon source and electron donor,
and nitrate as electron acceptor. Additionally, CDH is able to
catalyze nonphysiological asymmetric CꢀC bond formation.
Initially, the cross-benzoin reaction of benzaldehyde (4) and
pyruvate (5) (after decarboxylation) to result in the R-config-
ured 1-hydroxy-1-phenylpropan-2-one (PAC, 6, 98% ee) was
performed on an analytical scale (Scheme 1B).[6]
Thiamine diphosphate (ThDP)-dependent enzymes are in-
volved in a wide range of metabolic pathways and catalyze a
broad variety of reactions. Among these are oxidative and non-
oxidative decarboxylation and asymmetric CꢀC, CꢀO, CꢀS,
and CꢀN bond formation, as well as CꢀC bond cleavage.[1]
Prominent representatives include the enzymes transketolase,
pyruvate dehydrogenase, pyruvate decarboxylase, acetolactate
synthase, desoxyxylulose 5-phosphate synthase, benzaldehyde
lyase, and benzoylformate decarboxylase (BFD).[1b]
Until recently, CDH has been purified from its native source,
Azoarcus sp. strain 22Lin, by a multistep chromatographic pro-
tocol. Together with the cofactors ThDP, FAD, and MgII, the
enzyme monomer has
a theoretical molecular mass of
ThDP-dependent cyclohexane-1,2-dione hydrolase (CDH, EC
3.7.1.11) is the key enzyme of an anaerobic degradation path-
way of alicyclic alcohols. It catalyzes CꢀC bond cleavage of
cyclohexane-1,2-dione (1) to produce 6-oxohexanoic acid (2) as
the primary product, presumably[2] followed by oxidation of
the latter to adipic acid (3, Scheme 1A).[3–6] This degradation
pathway of a 1,2-diketone was discovered for the denitrifying
64.5 kDa.[3] Whereas SDS-PAGE shows a single protein band for
the monomer and size exclusion chromatography indicates
that CDH is a homodimer in solution, the crystal structure
shows a homotetramer with one noncovalently bound FAD
and one ThDP (bound to the protein with an MgII ion that is
coordinated to its diphosphate moiety) per monomer.[3–6]
The oligonucleotide and amino acid sequences of CDH do
not show pronounced similarities to related enzymes, despite
having typical cofactor-binding domains.[8] Interestingly, 1 is
also a substrate of ThDP-dependent YerE from Yersinia pseudo-
tuberculosis;[9] however, YerE does not display a-ketolase activi-
ty (observed with CDH for this substrate), but instead catalyzes
the addition of activated acetaldehyde to result in a tertiary
alcohol.[9] We anticipated that CDH could possess further dis-
tinctive features concerning the range of catalytic possibilities.
Herein, we describe the detailed characterization of CDH-cata-
lyzed asymmetric transformations, facilitated by heterologous
production of the enzyme. The results demonstrate that CDH
complements other well-known ThDP-dependent enzymes
with respect to substrate range and reaction scope.
[a] S. Loschonsky, S. Waltzer, Prof. Dr. M. Mꢀller
Institut fꢀr Pharmazeutische Wissenschaften
Albert-Ludwigs-Universitꢁt Freiburg
Albertstrasse 25, 79104 Freiburg (Germany)
E-mail: michael.mueller@pharmazie.uni-freiburg.de
[b] Dr. S. Fraas, Prof. Dr. P. M. H. Kroneck
Institut fꢀr Biologie, Universitꢁt Konstanz
Universitꢁtsstrasse 10, 78464 Konstanz (Germany)
[c] T. Wacker, Prof. Dr. S. L. A. Andrade
Institut fꢀr Biochemie and BIOSS Center for Biological Signalling Studies
Albert-Ludwigs-Universitꢁt Freiburg
Albertstrasse 21, 79104 Freiburg (Germany)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201300673.
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For optimal production and isolation of CDH, a synthetic
gene was designed with codon optimization for expression
with a C-terminal His6 tag from pET21a in Escherichia coli (see
the Supporting Information). E. coli BL21(DE3) cells were trans-
formed with the plasmid, grown in lysogeny broth (LB), and
gene expression was induced by isopropyl-b-d-thiogalactopyr-
anoside (IPTG). Recombinant CDH was purified by affinity chro-
matography (Ni-NTA). The purity of the protein was confirmed
by SDS-PAGE (see the Supporting Information), and the pres-
ence of the cofactor FAD was confirmed by UV/vis spectrosco-
py. Approximately 15 mg of pure CDH was obtained from 1 L
of culture. Although riboflavin supplement in heterologous
growth cultures has been reported to optimize the expression
of some FAD-dependent enzymes,[10] in the case of CDH we
did not observe any effect of riboflavin (up to 50 mgLꢀ1) on
the final yield of holoenzyme.
hydrophobic aromatic aldehydes in screening experiments on
the carboligase activity of CDH.
The scope and limitations of the CꢀC bond-formation capa-
bility of CDH were determined by testing 24 monosubstituted
benzaldehydes 7a,b,c–14a,b,c as putative acceptors and pyru-
vate (5) as donor in the formation of the corresponding PAC
derivatives 15a,b,c–22a,b,c (Table 1). Overall, in terms of con-
version and ee of the PAC products, benzaldehydes with one
electron-rich substituent (OH, OMe, Me) in the ortho, meta, or
para position (substrates 11–13) gave slightly better results
than 7–10 and 14 (electron-withdrawing groups; F, Cl, Br, I,
NO2). Benzaldehydes substituted in the para or meta positions
showed better conversion than the corresponding ortho-sub-
stituted derivatives.
Notably, CDH accepted hydroxy- and nitrobenzaldehydes 11
and 14, which are not (or only in rare cases)[12] known as sub-
strates of other ThDP-dependent enzymes. There was no con-
version with o-nitrobenzaldehyde (14a), which was thus the
only tested aromatic aldehyde not to be accepted by CDH.
Unexpectedly, CDH showed particularly low conversion of 2-
fluorobenzaldehyde (7a; see below).
All 18 PAC products with determined ee values (Table 1)
were assigned as R-configured, as judged by the appearance
of a negative band centered at 270–290 nm in circular dichro-
ism spectra.[13,14] All 18 products were highly enantioenriched,
with ee values in the range of 92–99%.
To determine the catalytic activity of recombinant CDH, both
cleavage of cyclohexane-1,2-dione (1, Scheme 1A) and the
cross-benzoin reaction of benzaldehyde (4) and pyruvate (5,
Scheme 1B) were examined. When 1 was incubated with CDH
in the absence of NAD+, the cleavage reaction stopped at 6-
oxohexanoic acid (2). Thus, the cleavage activity of CDH could
be determined unequivocally by proton NMR spectroscopy:
acidification of the aqueous enzymatic assay and extraction
with CDCl3 gave 2, which displayed a well-defined signal for its
aldehyde proton (dH-6 =9.78 ppm, triplet, J6,5 =1.5 Hz); further-
more, 1 seemed to exist exclusively as its 2,3-enol form (dH-3
=
As para-substituted and electron-rich benzaldehydes were
accepted best in terms of conversion, we applied sterically
more demanding aromatic aldehydes 23–29 in the cross-ben-
zoin reaction with pyruvate (Scheme 2).
6.15 ppm, triplet, J3,4 =4.6 Hz). The formation of the PAC prod-
uct (6) was monitored by GC/MS. Preliminary experiments sug-
gested that a 10 mm:25 mm ratio of 4 to 5 results in the high-
est conversion after 24 h. In the first few hours, formation of 6
showed first-order kinetics. In the linear region (0–4.7 h), the
specific activity of CDH was 9.5 mUmgꢀ1 purified protein (see
the Supporting Information). Conversion of 4 into (R)-PAC ((R)-
6) was 82% after 24 h, as determined by GC/MS; ee was 99%,
as determined by chiral-phase HPLC.
In general, these substrates were almost completely convert-
ed, and products 30–35 were obtained virtually enantiopure.
The reaction of 4-(tert-butyl)benzaldehyde (26!33, 98% con-
version) is noteworthy. Although the 2-hydroxy ketone 33 was
obtained with a relatively lower ee (92%), it should be empha-
sized that compounds bearing tert-butyl groups are notorious-
ly poor substrates in enzymatic transformations.[15] This is be-
cause of the high hydrophobicity of tert-butylated substrates
(thus low solubility in aqueous media) and the steric hindrance
imposed by the tert-butyl group. In our example, DMSO (20%
v/v) as the solubility promoter undoubtedly alleviated the solu-
bility challenge.
Thus, we confirmed that recombinant CDH shows the same
CꢀC bond-cleavage and CꢀC bond-formation activity as report-
ed for CDH purified from its native source, Azoarcus sp. strain
22Lin. Furthermore, it was shown that 20–30% (v/v) DMSO had
no negative effects on the conversion.[11] This allows the use of
Table 1. CDH-catalyzed conversion (%) of 7–14 and enantiomeric excess (% ee in brackets) for the formation of PAC derivatives 15–22.[a]
R
F
Cl
Br
I
OH
OMe
12
Me
13
NO2
14
substrate
product
7
15
8
16
9
17
10
18
11
19
20
21
22
ortho (a)
meta (b)
para (c)
5 (n.d.)
47 (98)
68 (96)
6 (n.d.)
34 (98)
82 (96)
24 (99)
49 (96)
69 (95)
45 (96)
51 (94)
81 (96)
19 (n.d.)
82 (99)
30 (98)
37 (99)
96 (92)
85 (98)
62 (99)
91 (98)
97 (96)
0 (–)
20 (n.d.)
5 (n.d.)
[a] Conditions: pyruvate (25 mm), aromatic aldehyde (10 mm), CDH (1 mgmLꢀ1), buffer A (50 mm MES, 1 mm MgSO4, 0.5 mm ThDP, pH 6.5), DMSO (20–
30%, v/v), 308C, 48 h; % conversion was determined by GC/MS. Enantiomeric excess was determined by chiral-phase HPLC; n.d. =not determined.
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experiment with 7a (a potential inhibitor), 4-ethylbenzalde-
hyde (23, one of the best acceptors), and pyruvate (5, donor).
In the absence of 7a, conversion of 23 to 1-(4-ethylphenyl)-
1-hydroxypropan-2-one (30) was 95% after 24 h (Table 2,
Table 2. Competition experiments with 2-fluorobenzaldehyde (7a), 4-eth-
ylbenzaldehyde (23), and pyruvate (5).[a]
Quantity [mm]
Product [%]
7a
23
5
15a
30
1
–
–
10
5
1
0.1
10
10
10
10
10
10
25
25
25
25
25
25
–
–
2
6
61
95
98
1
1
34
82
2[b]
3
4
5
6
Scheme 2. Screening sterically demanding aromatic aldehydes as acceptors
with pyruvate (5, donor) in the CDH-catalyzed cross-benzoin reaction. Condi-
tions as in Table 1.
n.d.[c]
[a] Conditions: pyruvate (25 mm), benzaldehydes 7a and 23 (see Table 2),
CDH (c=1 mgmLꢀ1), buffer A (see Table 1), DMSO (20–30% v/v), 308C,
24 h; % conversion (each value is an average from two independent en-
zymatic assays) was determined by GC/MS. [b] After 48 h. [c] Not detect-
ed.
Moreover, CDH used naphthaldehydes efficiently as acceptor
substrates, although the sterically hindered 1-naphthaldehyde
(29!36) showed only 36% conversion (>99% conversion of
the less hindered 2-naphthaldehyde (28!35)). Previously, 2-
naphthaldehyde (28) has been applied as a donor substrate in
BFD-catalyzed cross-benzoin condensation: in the presence of
solubilizing agents, only 34% conversion was obtained after
72 h.[16] In addition, 1-naphthaldehyde (29) was not accepted
at all by BFD, thus further highlighting the significance of our
results with CDH. The transformations of 4-(tert-butyl)benzalde-
hyde (26!33) and 2-naphthaldehyde (28!35) were also per-
formed on a semipreparative scale. The 2-hydroxy ketone
products 33 and 35 were obtained in 90% (28 mg) and 87%
(26 mg) isolated yields, respectively.
entry 1) and 98% after 48 h (entry 2). In the presence of equi-
molar amounts of 7a and 23 the conversion was just 1%
(entry 3); interestingly, 1-(2-fluorophenyl)-1-hydroxypropan-2-
one (15a) was detected in only trace amounts. Lowering the
7a:23 ratio revealed that 7a has a strong negative impact on
the formation of 1-(4-ethylphenyl)-1-hydroxypropan-2-one (30):
34% conversion of 23!30 when present as low as 10% rela-
tive to 23 (entry 5).
We then turned our attention to other aromatic aldehydes
possessing (highly) electron-withdrawing substituents. A po-
tential inhibitory effect on the formation of 30 was tested in
independent competition experiments under conditions analo-
gous to those listed in Table 2, entry 4. As anticipated, 2-chlor-
obenzaldehyde (8a), as well as 2,6-difluoro-, 2,4,5-trifluoro-,
and pentafluorobenzaldehyde, showed the same effect as 7a:
each of these benzaldehydes yielded ꢁ6% conversion into the
corresponding PAC products when incubated with pyruvate
(5), and inhibited the formation of 30 in the same manner as
7a (<2% conversion for 23!30). Detailed kinetic studies are
required to obtain a mechanistic explanation of these results
and to confirm the role of these electron-deficient benzalde-
hydes as potential inhibitors of the ligase activity of CDH.
In summary, we have described the heterologous expression
in E. coli and purification of recombinant His-tagged cyclohex-
ane-1,2-dione hydrolase (CDH). In addition to its physiological
CꢀC bond-cleavage activity, CDH catalyzes the asymmetric
cross-benzoin reaction of a broad variety of aromatic alde-
hydes and pyruvate (up to quantitative conversion, 92–99%
ee). In the case of the sterically demanding 4-(tert-butyl)benzal-
dehyde (26) and 2-naphthaldehyde (28), the respective 2-hy-
droxy ketone products (33 and 35) were obtained in high
yield. Notably, CDH accepts several benzaldehydes, such as hy-
In contrast to the broad acceptor substrate spectrum, the
donor substrate range of the CDH-catalyzed cross-benzoin re-
action with benzaldehyde (4) was, in effect, limited to pyruvate
(82% conversion). Aliphatic a-oxo acids larger than 2-oxobuta-
noic acid (1.3% conversion) were not accepted at all, and nei-
ther were 2-oxosuccinate, 2-oxoglutarate, or hydroxypyruvate
(the last substrate was tested with 4-ethylbenzaldehyde as
acceptor). In the absence of an aromatic acceptor aldehyde,
highly enantioenriched (S)-acetoin was formed by homocou-
pling of pyruvate.[17]
Analysis of the data in Table 1 with respect to the conversion
of ortho-substituted halobenzaldehydes reveals a trend that is
contrary to what was expected. On the basis of the steric
requirements and the electrophilicity of the halo substituents,
2-fluorobenzaldehyde (7a) should be accepted best as a sub-
strate, and 2-iodobenzaldehyde (10a) should be accepted
least. (This trend is regularly observed with other ThDP-depen-
dent enzymes.)[9,18] However, CDH catalysis displayed the oppo-
site outcome (Table 1). Accordingly, we hypothesized that 2-
fluorobenzaldehyde (7a) might act as an inhibitor of the car-
boligase activity of CDH. To test this, we set up a competition
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in buffer A (final total volume 15 mL) in a 15 mL Falcon tube. CDH
(1 mgmLꢀ1) was added, and the mixture was incubated at 308C
with shaking (400 rpm) for 24 h (for 28) or 48 h (for 26) until GC/
MS indicated 99% conversion. The reaction mixture was extracted
with MTBE (3ꢂ15 mL). The combined organic layers were dried
over MgSO4, and the volatiles were removed under reduced pres-
sure. The crude product was isolated with an Isolera preparative
flash purification system (Biotage AB, Uppsala, Sweden).
droxy- and nitrobenzaldehydes; these are not (or only in rare
cases) known as substrates of other ThDP-dependent enzymes.
An inhibitory effect of benzaldehydes with electron-withdraw-
ing substituents on the carboligase activity was observed.
Hence, CDH expands the range of well-known ThDP-depen-
dent enzymes suitable as catalysts in asymmetric carboligation
reactions. The straightforward production and stability of this
enzyme, as well as its unique behavior as a powerful multi-
functional catalyst, make it an interesting starting point for
synthetic applications and mechanistic studies.
(R)-1-(4-tert-Butylphenyl)-1-hydroxypropan-2-one (33) was obtained
in 90% yield (28 mg). (R)-1-Hydroxy-1-(naphthalen-2-yl)propan-2-
one (35) was obtained in 87% yield (26 mg).
Experimental Section
Acknowledgements
Cloning: The synthetic, codon-optimized, cdh gene from Azoarcus
sp. 22Lin (GeneArt, Life Technologies) was cloned into a pET21a
vector (Novagen), between NdeI and XhoI restriction sites. Omis-
sion of the gene’s stop codon allowed expression with the plas-
mid-encoded C-terminal His6 tag.
This work has been supported by the German Research Founda-
tion (DFG) through grants SPP 1319 (to P.M.H.K), FOR 1296 (to
M.M.), and AN 676/1 (to S.L.A.A.).
Expression and purification of CDH: For the production of His-
tagged CDH, E. coli BL21(DE3) was transformed with pET21a carry-
ing the cdh gene. A preculture was grown in LB (7 mL) with ampi-
cillin (100 mgmLꢀ1) at 378C for 24 h with shaking (140 rpm). From
this, an aliquot was incubated (258C, 130 rpm) in fresh LB (500 mL)
with ampicillin (100 mgmLꢀ1). Upon reaching OD600 =0.6–0.7 (ca.
5.5 h), IPTG (0.02 mm) was added to induce gene expression. After
17–24 h at 258C (130 rpm), cells were harvested by centrifugation
(5200g, 40 min, 48C). From the 500 mL culture, 2.5–3.0 g of cells
were obtained. The cell pellet was resuspended in buffer A (15 mL;
MES (2-(N-morpholino)ethanesulfonic acid, 50 mm, pH 6.5), MgSO4
(1 mm), ThDP (0.5 mm)) and lysed by a French press. Cell debris
was removed by centrifugation (6000g, 45 min, 48C), and the
lysate (20 mL) was incubated with Ni-NTA resin (2 mL) at 08C for
1 h. After washing with buffer A containing imidazole (20 then
50 mm), recombinant CDH was eluted in buffer A containing imi-
dazole (300 mm). The combined elution fractions were desalted by
gel-permeation chromatography in buffer A in a Sephadex column
(GE Healthcare). The purity of the protein was confirmed by SDS-
PAGE (see the Supporting Information). Approximately 15 mg puri-
fied protein was obtained from 1 L cell culture.
Keywords: asymmetric catalysis
carboligation · enzyme catalysis · ThDP
·
CꢀC bond-formation
·
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and pyruvate (analytical scale): Enzymatic reactions on an analyti-
cal scale were performed in buffer A (1.5 mL) containing DMSO
(20–30% v/v). The aromatic aldehyde (final concentration 10 mm)
was applied as a DMSO solution; sodium pyruvate (25 mm) and
the enzyme (1 mgmLꢀ1) were added. All reactions were conducted
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CDH-catalyzed synthesis of 33 and 35 (semipreparative scale): A
solution of 4-(tert-butyl)benzaldehyde (26, 25.1 mL, 24.3 mg,
0.150 mmol, final concentration in the reaction assay 10 mm) in
DMSO (3.75 mL, final concentration 25%, v/v) or 2-naphthaldehyde
(28, 23.4 mg, 0.150 mmol, final concentration 10 mm) in DMSO
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Received: October 22, 2013
Published online on January 16, 2014
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ChemBioChem 2014, 15, 389 – 392 392