Angewandte
Chemie
[
5]
2
2Lin, CDH catalyzes the CꢀC bond cleavage of cyclohex-
atom of 4 in the active site were replaced, while in CDH-
[6]
ane-1,2-dione (4) to 6-oxohexanoic acid (5; Scheme 1B).
H76A/Q116A, the amino acids coordinating the other oxygen
atom of 4 in the active site were changed. The variants were
prepared with a C-terminal hexahistidine tag, expressed in
E. coli BL21(DE3) cells and purified by affinity chromatog-
raphy (see the Supporting Information).
The CꢀC bond cleavage is assumed to be initiated by the
attack of the ThDP ylide on the C=O bond of the mono-
[
7]
hydrate 6 of 1,2-diketone 4 to form the ThDP adduct 7
Scheme 1B).
[6b]
(
Compound 7 can also be regarded as
[3]
a tetrahedral intermediate which breaks down to the carbox-
ylic acid 8. Protonation of the enamine moiety of 8 results in 9
and the subsequent elimination of ThDP furnishes the oxo
Wild-type (wt) CDH and the purified variants were
screened towards 1) the formation of PAC (3) and 2) the 1,2-
diketone cleavage of 4 (Table 1). Three variants (H76A,
[
6b]
acid 5. A seemingly related CꢀC bond cleavage of 3,4,5-
trihydroxycyclohexane-1,2-dione to 5-deoxy-d-glucuronic
[
a]
[b]
Table 1: CꢀC bond-formation and CꢀC bond-cleavage reactivity of
[
8]
acid is catalyzed by IolD from Bacillus subtilis. However,
CDH and IolD are not homologues and no (significant)
sequence homologue of CDH has been identified in the
TEED database. Thus, CDH represents a unique enzyme
with respect to reactivity and amino acid sequence.
wt-CDH and six variants.
[9]
Although cyclohexane-1,2-dione (4) is a substrate of a
CꢀC bond-cleavage reaction catalyzed by CDH (!5), CDH
is unable to catalyze CꢀC bond formation (carboligation)
using pyruvate (1) as acyl anion donor and 4 as the acceptor
(
Scheme 1C). Conversely, ThDP-dependent YerE from Yer-
sinia pseudotuberculosis utilizes 4 as an acceptor in a carbo-
ligation reaction with pyruvate (1) to form the tertiary alcohol
[
c]
[d]
CDH
variant
Conversion [%] (ee [%])
1+2!3
[10]
4!5
1
0 (Scheme 1C). In fact, YerE was the first recombinant
enzyme found to catalyze cross-benzoin condensations with
wt
N484A
H28A
H76A
Q116A
H28A/N484A
H76A/Q116A
98 (99, R)
18 (99, R)
90
48
78
3
3
17
9
[
10]
nonactivated ketones as acceptors. Apart from YerE, very
few ThDP-dependent enzymes have been shown to accept
ketones as acceptor substrates. Recently, Jiang et al. reported
the addition of pyruvate to acetone in a reaction catalyzed by
12 (>99, R)
[
e]
3 (n.d.)
0 (n.d.)
[
e]
73 (>99, R)
acetoin:2,6-dichlorophenolindophenol
oxidoreductase
[e]
0 (n.d.)
[
11]
(
AcoAB) from Bacillus subtilis.
1,2-Diketones are also
[a] Conditions: pyruvate (25 mm), benzaldehyde (10 mm), CDH variant
substrates of an as yet uncharacterized enzyme from Bacillus
licheniformis, which catalyzes cleavage–readdition
ꢀ1
(1 mgmL ), buffer (50 mm MES, 1 mm MgSO , 0.5 mm ThDP, pH 6.5),
4
a
308C, 48 h. [b] Conditions: cyclohexane-1,2-dione (25 mm), CDH variant
[
12]
ꢀ1
sequence.
(1 mgmL ), buffer (50 mm MES, 1 mm MgSO , 0.5 mm ThDP, pH 6.5),
4
1
Using both site-directed and saturation mutagenesis,
ThDP-dependent enzymes have been engineered to provide
finely tuned catalytic properties, with a focus on increased
308C, 48 h. [c] Determined by H NMR spectroscopy (400 MHz, CDCl ).
[d] Determined by HPLC on a chiral stationary phase. [e] Not deter-
mined. MES=2-(N-morpholino)ethanesulfonic acid.
3
[
13]
substrate range and enhanced stereospecificity.
Yet, to
date, no variants have been generated that are able to use
ketones as the acceptor substrates in carboligation reactions.
In this report, we present protein engineering experiments
designed to selectively knock out either 1) the CꢀC bond-
formation reactivity or 2) the CꢀC bond-cleavage reactivity
Q116A, and H76A/Q116A) proved to be essentially inactive,
while two other variants showed a selective reduction of one
of the two activities. The H28A variant showed an eightfold
decrease in the formation of PAC (12%), but 1,2-diketone
cleavage was nearly unaffected (78% conversion). The
double variant H28A/N484A showed acceptable formation
of PAC (73%), but conversion toward the cleavage product
was decreased by a factor of five (17% conversion). Regard-
less of the mutation, the PAC product (3) of every active
variant had (R)-configuration (ꢁ 99% ee).
of CDH. Furthermore, we present a CDH variant whose
catalytic properties have been fundamentally changed such
that the 1,2-diketone 4, which originally served as a substrate
for CDH-catalyzed CꢀC bond cleavage, is now accepted as
a substrate for CꢀC bond formation.
Steinbach et al. reported the crystal structure of native
CDH, showing it to be a homotetrameric protein with one
Overall, the H28A/N484A variant showed considerably
reduced relative cleavage reactivity with its physiological
substrate (4!5, 17% conversion), but still catalyzed CꢀC
[
6c,d]
FAD and one ThDP per monomer.
Crystals soaked with
cyclohexane-1,2-dione (4) showed that, in addition to ThDP,
four amino acids (N484, from one monomer, and H28, H76,
and Q116 from a second monomer) directly interact with the
bond formation using pyruvate as a donor (1 + 2!3). There-
fore, we speculated that this variant might catalyze a CꢀC
[
6c,d]
carbonyl groups of 4 at the active site.
role of these residues, we prepared four single variants
H28A, H76A, Q116A, and N484A), as well as two double
To investigate the
bond-forming reaction, again with pyruvate (1) as a donor,
but now using 4 as the acceptor. To date, the only known
enzyme to accept 4 in such a reaction is YerE (32%
(
[10]
variants, H28A/N484A and H76A/Q116A. In CDH-H28A/
N484A, the active-site residues coordinating one oxygen
conversion, 22% ee; Scheme 1). We were pleased to see
that the H28A/N484A variant of CDH showed a comparable
Angew. Chem. Int. Ed. 2014, 53, 14402 –14406
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Loschonsky, Sabrina
