Origins of stereoselectivity in evolved ketoreductases
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Elizabeth L. Noey , Nidhi Tibrewal , Gonzalo Jiménez-Osés , Sílvia Osuna , Jiyong Park , Carly M. Bond , Duilio Cascio ,
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Jack Liang , Xiyun Zhang , Gjalt W. Huisman , Yi Tang
, and Kendall N. Houk
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Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095; Department of Chemical and Biomolecular Engineering,
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University of California, Los Angeles, CA 90095; Molecular Biology Institute, University of California, Los Angeles, CA 90095; and Codexis Inc., Redwood
City, CA 94063
Contributed by Kendall N. Houk, September 21, 2015 (sent for review April 28, 2015; reviewed by William A. Goddard III, Donald Hilvert, and Iñaki Tuñón)
Mutants of Lactobacillus kefir short-chain alcohol dehydrogenase,
used here as ketoreductases (KREDs), enantioselectively reduce the
pharmaceutically relevant substrates 3-thiacyclopentanone and
alkoxide formed on hydride transfer from NADPH (10, 11). The
alcohol is then liberated through a proton relay involving Tyr156,
the cofactors’ ribofunarose, Lys160, a backbone carbonyl, and
water. When the substrate’s substituents differ in size, the smaller
(R ) and larger (R ) substituents bind to the small and large binding
pockets, respectively (Fig. 2B) (12). It has been proposed that Tyr190
prevents binding of the large group in the small binding pocket (13).
These enzymes are tetramers in their crystallographic and
loop spanning roughly residues 190–210, the most variable por-
tion of SDRs, which has been implicated as crucial in determining
the stereoselectivity of KREDs (14). In its closed conformation,
this motif flanks one side of the active site and closes around the
bound cofactor and substrate (11).
A plot of the enantioselectivities for the reduction of 1 and 2
given by 175 evolved KRED variants developed by Codexis is
shown in Fig. 3. Despite the broad distribution of S/R enantiomeric
ratios (erS/R) observed, some of these enzymes are highly enantio-
selective and are able to distinguish between a sulfur or oxygen
atom and a methylene group, both located β to the reacting car-
bonyl group. Because the WT enzyme was evolved especially for
3-oxacyclopentanone. These substrates differ by only the heteroatom
(S or O) in the ring, but the KRED mutants reduce them with differ-
S
L
ent enantioselectivities. Kinetic studies show that these enzymes
are more efficient with 3-thiacyclopentanone than with 3-oxacyclo-
+
pentanone. X-ray crystal structures of apo- and NADP -bound selected
mutants show that the substrate-binding loop conformational prefer-
ences are modified by these mutations. Quantum mechanical calcula-
tions and molecular dynamics (MD) simulations are used to investigate
the mechanism of reduction by the enzyme. We have developed an
MD-based method for studying the diastereomeric transition state com-
plexes and rationalize different enantiomeric ratios. This method, which
probes the stability of the catalytic arrangement within the theozyme,
shows a correlation between the relative fractions of catalytically com-
petent poses for the enantiomeric reductions and the experimental
enantiomeric ratio. Some mutations, such as A94F and Y190F, induce
conformational changes in the active site that enlarge the small binding
pocket, facilitating accommodation of the larger S atom in this region
and enhancing S-selectivity with 3-thiacyclopentanone. In contrast, in
the E145S mutant and the final variant evolved for large-scale produc-
tion of the intermediate for the antibiotic sulopenem, R-selectivity is
promoted by shrinking the small binding pocket, thereby destabilizing
the pro-S orientation.
3
-thiacylopentanone, the variants are not equally selective for
both substrates. Nearly all of the mutants, including the WT, give
Significance
directed evolution crystallographic structures
| molecular dynamics
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theozyme enantioselectivity
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Ketoreductases are the most commonly used enzymes in in-
dustrial pharmaceutical synthesis. We investigated the nature
of enantioselectivity in closely related mutant ketoreductases
that reduce almost-symmetrical 3-oxacyclopentanone and
iocatalysis is a common method of stereoselective ketone re-
duction (1). This approach often replaces multistep syntheses
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and uses renewable, biodegradable, and nontoxic reagents and
mild conditions (2). Ketoreductases (KREDs), the most commonly
used enzymes in industrial pharmaceutical synthesis (3), reduce a
wide range of ketones to alcohols with high chemoselectivity and
stereoselectivity. These enzymes have been engineered to synthe-
size alcohols as intermediates for the production of atorvastatin
3-thiacyclopentanone, which are difficult to reduce enantiose-
lectively by other means. We present the efficiencies of select
variants and their crystallographic structures. Our experimental
and theoretical studies reveal how mutations modulate the
stereoselectivity of the reduction. Molecular dynamics simula-
tions of the Michaelis–Menten and transition state-bound com-
plexes were used to rationalize the observed stereochemical
outcomes. We discovered that the closed conformation of the
flexible substrate binding loop is likely the catalytically active one
imparting the stereochemical preferences. Our molecular dy-
namics approach reveals how each enzyme stabilizes the di-
astereomeric transition structures by altering the active site size.
(
Lipitor), montelukast (Singulair), and atazanavir (Reyetaz) (4).
Small and almost symmetrical ketones, such as prochiral cyclo-
pentanones, are attractive substrates that are difficult to reduce
asymmetrically by chemical methods (5, 6). In particular, the
enantiopure chiral alcohols derived from 3-oxacyclopentanone (1)
and 3-thiacyclopentanone (2) are used in the synthesis of the
pharmaceutical agents fosamprenavir and sulopenem, respectively
(Fig. 1). Through a directed evolution (DE) program, Codexis, Inc.
Author contributions: D.C., X.Z., G.W.H., Y.T., and K.N.H. designed research; E.L.N., N.T.,
G.J.-O., S.O., J.P., C.M.B., and J.L. performed research; E.L.N., N.T., G.J.-O., S.O., J.P., C.M.B.,
and J.L. analyzed data; and E.L.N., N.T., G.J.-O., S.O., J.P., C.M.B., D.C., J.L., X.Z., G.W.H., Y.T.,
and K.N.H. wrote the paper.
engineered a KRED obtained from Lactobacillus kefir for the re-
duction of 3-thiacyclopentanone (2) for the large-scale production
of the antibiotic sulopenem. L. kefir KRED (WT) belongs to the
short-chain dehydrogenase/reductase (SDR) family (7, 8). Via DE,
a variant containing 10 mutations (so-called Sph) was obtained and
used for large-scale synthesis of (R)-3-thiacyclopentanol.
Reviewers: W.A.G., California Institute of Technology; D.H., ETH Zurich; and I.T.,
Universitat de València.
The authors declare no conflict of interest.
Data deposition: The coordinates of the X-ray structures of variants apo WT, holo WT, apo
Sph and related KREDs use the NADPH cofactor as hydride
reductant. In the industrial process, NADPH is regenerated by
oxidation of glucose to glucono-1,5-lactone with glucose de-
hydrogenase. The proposed mechanism for carbonyl reduction
and subsequent protonation of the intermediate (9) is shown in
Fig. 2A. In L. kefir numbering, Ser143 and Tyr156 stabilize the
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PNAS Early Edition
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