Origins of stereoselectivity in evolved ketoreductases

Article abstract of DOI:10.1073/pnas.1507910112

Mutants of Lactobacillus kefir short-chain alcohol dehydrogenase, used here as ketoreductases (KREDs), enantioselectively reduce the pharmaceutically relevant substrates 3-thiacyclopentanone and 3-oxacyclopentanone. These substrates differ by only the heteroatom (S or O) in the ring, but the KRED mutants reduce them with different enantioselectivities. Kinetic studies show that these enzymes are more efficient with 3-thiacyclopentanone than with 3-oxacyclopentanone. X-ray crystal structures of apo- and NADP⁺-bound selected mutants show that the substrate-binding loop conformational preferences are modified by these mutations. Quantum mechanical calculations 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 complexes 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 competent 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 production of the intermediate for the antibiotic sulopenem, R-selectivity is promoted by shrinking the small binding pocket, thereby destabilizing the pro-S orientation.

Full text of DOI:10.1073/pnas.1507910112

Origins of stereoselectivity in evolved ketoreductases
a
b
a
a
a
b
c
Elizabeth L. Noey , Nidhi Tibrewal , Gonzalo Jiménez-Osés , Sílvia Osuna , Jiyong Park , Carly M. Bond , Duilio Cascio ,
d
d,1
d,1
a,b,1
a,b,1
Jack Liang , Xiyun Zhang , Gjalt W. Huisman , Yi Tang
, and Kendall N. Houk
a
b
Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095; Department of Chemical and Biomolecular Engineering,
c
d
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
catalytically active forms, although they are purified as monomers
(SI Appendix, Fig. S1). They feature a flexible substrate-binding
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
|
|
theozyme enantioselectivity
|
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
B
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
E145S, and apo A94F have been deposited in the Protein Data Bank, www.rcsb.org/pdb/
home/home.do (PDB ID codes 4RF4, 4RF2, 4RF5, and 4RF3, respectively).
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
1
To whom correspondence may be addressed. Email: zhangxiy@hotmail.com, Gjalt.
Huisman@codexis.com, yitang@ucla.edu, or houk@chem.ucla.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1507910112/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1507910112
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methanol, phenol, and methylammonium, respectively. The
calculated transition states (TSs) for the reduction of ketones 1
and 2 are shown in Fig. 4. As shown by their activation barriers,
3
-thiacyclopentanone 2 is reduced more easily than 1 by ∼2 kcal
−1
mol , consistent with the higher efficiencies measured for 2 with
all enzymes. This higher reactivity of 2 would not be expected if
only inductive effects—which are greater for 3-oxacyclopentanone
1
—were important. Conversely, a distortion/interaction analy-
sis (15) showed that 3-thiacyclopentanone is easier to distort
into the TS geometry, presumably owing to the longer C–S
bonds (1.85 Å, compared with 1.43 Å in C–O) and consequent
greater flexibility of the five-membered ring in 2 (SI Appendix,
Table S4).
In this highly simplified model, the calculated stereoselectivity
is induced by the restricted positions of the cofactor and catalytic
groups. Nevertheless, remarkably good agreement with the enan-
Fig. 1. Prochiral cyclopentanones 1 and 2 and their use in the synthesis of
two pharmaceuticals.
tioselectivities measured for the WT with 1 (S-selective; erS/R
.4) and 2 (R-selective; erS/R = 0.1), was obtained. These calcula-
tions reveal the intrinsic preferences for formation of the (R)- or
S)-cyclopentanols for each starting ketone when all of the cat-
=
5
(S)-3-oxacyclopentanol from 1. This enantioselectivity agrees with a
(
qualitative model by which the 3-oxa and 4-methylene groups bind
to the small and large binding pockets, respectively; however, the
enantioselectivity in the reduction of 2 ranges from erR/S ∼50 to
erS/R ∼10. These data indicate that in such small substrates, the
typical distinction between “large” and “small” substituents is far
from obvious.
alytic groups are arranged optimally. In fact, the calculated po-
sitions of the cofactor and catalytic side chains overlay almost
perfectly with those in the X-ray structure of the WT KRED in
+
the holo state (i.e., NADP -bound; SI Appendix, Fig. S4), dem-
onstrating the high preorganization of the active site achieved by
natural evolution to stabilize the TS.
The slight structural differences between ketones 1 and 2 were
analyzed in terms of computed molar volumes and electrostatic
potentials (SI Appendix, Figs. S2 and S3). O is slightly smaller but
Analysis of X-Ray Structures. Five crystal structures of L. kefir KRED
variants were obtained, four of them in the apo state (WT, E145S
and A94F single mutants, and the final variant Sph), and the WT in
the holo state, all crystallized as tetramers (Fig. 5A). Sph has 10
mutations, including two in the small binding pocket (E145S and
N157T), two in the large binding pocket (A94T and S96P), two in
considerably more polar than a CH group. S is closer in size and
2
2
polarity (slightly larger and more polar) to a CH . Remarkably, the
enzymes produced during DE—even single or double mutants—do
recognize these very subtle differences, promoting different levels
of enantioselectivity. To understand the origins of this exquisite
stereocontrol achieved by evolved KREDs in the reduction of these
small, almost symmetrical substrates, we performed crystallographic,
activity, and computational studies of the WT, selected single mu-
tants showing the largest differences in enantioselectivity, and the
most evolved variant, Sph.
1
0
the substrate-binding loop (P194N and M206Q), two in α-helix E
(
R108H and G117S), one in α-helix G (I223V), and one more in a
loop (G7S) (SI Appendix, Table S7). The side chain conformations
and hydrogen bonds established by these residues are obviously
different in the Sph and WT crystal structures, but their backbone
structures are very similar, with the exception of the P194N mu-
tation, which results in a less-ordered helix turn in the substrate-
binding loop in Sph. Further structural details are provided in SI
Appendix, Table S5 and Figs. S5–S12.
Results and Discussion
Enzymatic Activity and Theozyme Calculations. The catalytic efficiencies
(kcat/K ), enantiomeric ratios (erS/R), and associated differences in
M
‡
the activation barriers at 25 °C (ΔΔG S/R), assuming a rapid
equilibration between pro-R and pro-S Michaelis–Menten com-
plexes (i.e., Curtin–Hammett conditions), are summarized in Table 1
and in SI Appendix, Table S1. As these data show, the WT is the
most efficient of these enzymes, given that the evolution program
aimed to enhance enantioselectivity (>99% enantiomeric excess) in
this already active enzyme. Moreover, because of the evolutionary
pressure exerted toward 3-thiacyclopentanone, all of the variants
are systematically better catalysts for reduction of 2 than of 1; the
two more active and R-selective variants for 2 (E145S single mutant
and Sph) are poorly S-selective and ∼200 times less active for 1. The
decreased rate of reduction of 2 with A94F and Y190F, which favor
‡
formation of the S-enantiomer (er >1 and negative ΔΔG S/R),
shows a significantly reduced rate of formation of the normally
preferred R-enantiomer by these point mutations.
We assume that these differences in stereoselectivity emerge
mainly from the chemical step (related to kcat), which was the
main focus of our study, although contributions from other steps
Fig. 2. (A) Reduction (a) and alkoxide protonation (b) steps in KREDs. The
NADPH cofactor (in pink) is abbreviated, the substrate is in purple, and the
catalytic residues are in teal. The reactive hydride and the hydrogens pro-
posed to be involved in the proton relay are highlighted in red and blue,
respectively. (B) X-ray structure of holo L. kefir WT with acetophenone (in
purple) docked in. The cofactor (in pink) and residues in the active site are
shown as sticks. The catalytic triad (Ser143-Tyr156-Lys160) is in blue, and the
residues along the small and large binding pockets are shown in green and
dark blue, respectively.
M
in the catalytic cycle (i.e., substrate binding, related to K ) cannot
be ruled out.
Enzymatic reduction was modeled quantum mechanically (Ma-
terials and Methods), using an abbreviated quantum mechanical
(
QM) model of the active site. The NADPH cofactor was truncated
to a nicotinamide-ribofuranose complex, and the side chains of
the catalytic Ser143, Tyr156, and Lys160 were abbreviated to
2
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Noey et al.

Another minor difference observed in these structures is the
different hydrogen bond and crystallographic water patterns
exhibited by different variants for the noncatalytic Tyr190-Glu145-
Asp150 triad. As shown in Fig. 5 C–F, the bridging water molecule
trapped within this motif in the WT (Fig. 5C), is displaced by the
hydrophobic A94F mutation present in both the single mutant (Fig.
5
D) and the final Sph (Fig. 5F). In contrast, the E145S mutation
(
Fig. 5E) mimics the WT, in that two additional water molecules are
cocrystallized to maintain a similar distribution of oxygen atoms.
Molecular Dynamics Simulations of Enzyme-Substrate Complexes in
Water. Given the lack of conclusive structural information on the
effects of DE mutations on the variable catalytic profiles
exhibited by the KRED variants, we analyzed the same proteins
through molecular dynamics (MD) simulations in explicit water
starting from the available crystal structures (Materials and
Methods). These simulations predict the shape of the active site,
including the conformational states of the enzyme.
We first ran a series of unrestrained 100-ns trajectories of the
2+
Mg -bound tetramers with the cofactor (NADPH) and substrates
(1 or 2) docked in the active site. Whereas the cofactor stays
bound to the active site through multiple polar interactions, the
very small substrates are usually unable to maintain a productive
orientation in the active site, and in many cases the substrates
diffuse to the solvent after a short simulation time (∼25 ns). As
expected, the substrates are less mobile when the binding loop,
which is the most dynamic region of the protein, is in a closed
conformation. The closed-to-open transitions usually occur within
Fig. 3. Logarithmic graph of the experimental erS/R obtained by KRED-evolved
variants for 3-oxacyclopentanol (horizontal axis) and 3-thiacyclopentanol (verti-
cal axis). The values measured for selected variants (WT, A94F, Y190F, E145S, and
Sph) and calculated for simplified QM models are highlighted.
Of note, the structures of the holo WT and apo final mutant Sph
are nearly identical (rmsd over all Cα, 0.35 Å; a per-residue
comparison is shown in SI Appendix, Fig. S5) in both the protein
backbone and the active site, a situation that we have described
previously for another evolved enzyme also bearing a Ser-Tyr-Lys
catalytic triad (16). It is frequently observed that in enzymes with
such highly preorganized active sites based on complex hydrogen
bond networks, DE does not perturb the optimized catalytic ar-
∼20 ns. When the loop is closed, residues Leu199 and Leu195 in
this region establish van der Waals contacts with Ile93, Ala/Phe94,
and Ser96 in the binding pocket, with Cα–Cα distances of ∼7–10 Å
depending on the mutant; in the open conformation, these dis-
tances fluctuate within 12–18 Å. (SI Appendix, Figs. S15–S18 show
open/closed population distributions.) Thus, the conformational
rangement, at least in the solid state, and that the reasons for states of this loop were monitored and classified according to the
enhanced catalysis must be sought outside the active site.
distance between the Cα of Leu195 and that of Ala94 (Fig. 6A).
The results of per-residue fluctuation analysis are shown in SI
Appendix, Figs. S13 and S14.
Indeed, the most noticeable differences among these crystal
structures lie in the conformation of the substrate-binding loop:
either cofactor binding or DE mutations favor the closed con-
formation, whereas in the rest of the structures, the loop is more
open (Fig. 5B). In the structure of the A94F single mutant, the
active site is less solvent-exposed, and Phe94 makes a van der
Waals contact with Leu195 on the substrate-binding loop; this
promotes a slightly more closed conformation of the latter. This
is significant, because this mutation alone decreases the re-
activity and reverses the enantioselectivity in the reduction of
We found that the fraction of the simulation time during which
the loop is closed for each mutant correlates well with the experi-
mental relative activation barriers for the pro-R and pro-S ketone
‡
reduction pathways (ΔΔG S-R), especially for 3-thiacyclopentanone
2
, for which the enzymes were evolved (Fig. 6B). These results
confirm the key role of the binding loop structure and conforma-
tional dynamics in the activity and stereochemical outcome of the
reduction reactions (14). This was also demonstrated by the crystal
structures described above. Nevertheless, this seemingly very good
correlation should not be overinterpreted, given the difficulty of
clearly distinguishing between open and closed conformations in
some very flexible variants, such as Sph (SI Appendix, Fig. S18).
The specific mechanism through which the closed loop induces
asymmetry is not apparent. The closed conformation of the loop
3
-thiacyclopentanone with respect to the WT. Taken together,
these observations suggest that not only the structure, but also
the conformational dynamics, of the substrate-binding loop may
be related to the observed reactivity and stereoselectivity of the
reduction (see the discussion on molecular dynamics below).
‡
Table 1. erS/R, ΔΔG S-R, and kcat/K
M
values determined experimentally for 3-oxacyclopentanol and
3
-thiacyclopentanol obtained by KRED variants from ketones 1 and 2, respectively
3
-oxacyclopentanol (from 1)
3-thiacyclopentanol (from 2)
ΔΔG S-R, kcal mol−1
‡
k
cat/K
, mM min−1
-1
erS/R
ΔΔG S-R, kcal mol−1
‡
k
cat/K
, mM min−1
-1
Variant
erS/R
M
M
WT
4.2
27.5
9.3
4.2
−0.85
−1.95
−1.31
−0.84
−0.14
0.025
0.005
ND
0.003
0.004
0.2
2.6
1.6
0.02
0.004*
0.98
−0.55
−0.26
2.34
1.460
0.016
ND
0.772
0.703
A94F
Y190F
E145S
Sph
,†
1.3*
3.33
ND, not determined.
Measured under the experimental conditions described in SI Appendix, Fig. S33.
Obtained for a related variant lacking the P194N mutation.
*
†
Noey et al.
PNAS Early Edition
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pro-R and pro-S TSs, we developed a new MD-based methodology
using the QM geometries of the theozymes.
Restricted MD Simulations with the Theozyme in Water. Following
Pauling’s postulate, it is now generally accepted that enzymes are
complementary in structure to the TS of the reaction that they
catalyze (17). In accordance with this idea, we used MD simula-
tions to provide a detailed description of the active site near the
reduction TSs for selected DE variants (16, 18). The activated
complexes were built into the protein structure from the optimal
QM geometries and atomic partial charges of the diastereomeric
theozymes described above for substrates 1 and 2 (Fig. 4), fol-
lowing a similar protocol as described in the literature (19). The
distances of the breaking CNADPH–H and forming Csubstrate–H
bonds and the relative orientation of cofactor and substrates (pro-S
or pro-R) were fixed by defining special TS parameters, and the
systems were simulated for 160 ns in explicit water (Materials and
Methods and SI Appendix, Materials and Methods).
‡
For each of the four diastereomeric TS complexes—(R)-1 -,
‡
‡
‡
(
S)-1 , (R)-2 , and (S)-2 —the ability of each of the enzymes to
maintain productive catalytic contacts with the substrate and co-
factor along the simulations was analyzed by monitoring the four
distances shown in Fig. 8A. These involve the interaction between
hydrogen bond donors Ser143/Tyr156 and the developing alkoxide
in the substrate (distances a and b), and between Tyr156/Lys160 and
the cofactor ribofuranose (distances b and c) (SI Appendix, Table S8
presents average values). For each simulation step, the geometry
was considered catalytically competent—i.e., to adequately lower
the free energy of activation so that reaction occurs—only when all
of the selected distances were simultaneously lower than an arbi-
trary value similar to that calculated for the theozymes (3.4 Å) (SI
Appendix, Figs. S20 and S21 provide further details on the influence
of this threshold value). This is an indirect way of estimating the
number of reactive events occurring in a certain period, i.e., the rate
constant for a particular reaction pathway. In these circumstances,
and assuming rapidly equilibrating diastereoisomeric Michaelis–
Menten complexes (i.e., Curtin–Hammett conditions), the relative
populations of the reactive poses derived from the restrained pro-S
and pro-R trajectories (calc. erS/R) can be compared with the ex-
perimental enantiomer ratios measured for each prochiral ketone
Fig. 4. Transition structures leading to (R)-3-oxacyclopentanol (A), (S)-
-oxacyclopentanol (B), (R)-3-thiacyclopentanol (C), and (S)-3-thiacyclopentanol
D), calculated with CPCM(H O)/M062X/6–311+G(d,p)//TPSS/6–31G(d). Activation
3
(
2
‡
−1
free energies (ΔG ) are given in kcal mol ; distances, in Å.
certainly increases the substrate-binding affinity and creates a
more packed, reactive, and stereoselective environment in the
active site; however, if the open conformation cannot be achieved,
then certain substrates might be locked into nonproductive ori-
entations, or catalyst turnover may be decreased by slow substrate
binding and/or product release. This might be the case for the
A94F single mutant, for which the reduction of 2 is much slower
and with opposite stereoselectivity (S-selective) compared with WT
(
R-selective). With this variant, the substrate-binding loop always
(Table 1 and SI Appendix, Table S1).
remains in the closed conformation (Fig. 7), and the pro-R reaction
Because a certain population of the closed conformation of the
mobile substrate-binding loop is required for enantioselective ca-
R channel has an opposite effect on substrate 1, for which the WT is talysis, only those snapshots presenting the loop in a closed con-
pathway is significantly slowed. In contrast, this blocking of the pro-
already S-selective, further enhancing S-selectivity.
Careful inspection of the MD trajectory of the A94F single
formation (i.e., <10 Å distance between L195 and A/F94 Cα) were
used to derive the calculated er values. As shown in Fig. 8B,
S/R
mutant in complex with NADPH and 3-thiacyclopentanone 2 cor- these estimates correlate roughly with the experimental observa-
roborated this hypothesis. Throughout the entire simulation time,
the substrate is sequestered in a nonproductive orientation imposed
by four densely packed hydrophobic residues (Leu195 and Phe94,
both involved in the loop closing, along with Tyr190 and catalytic
Tyr156) (Fig. 7 A and B). This confined environment orients the
cyclopentanone ring perpendicular to the cofactor, precluding the
hydride transfer even though catalytic Ser143 and Lys160 are per-
manently hydrogen-bonded to the substrate carbonyl and NADPH
ribose, respectively; the other catalytic residue, Tyr156, stays close
to the substrate carbonyl but cannot hydrogen-bond to it (Fig. 7C).
On the other hand, the highly R-selective and more reactive
E145S and Sph variants sample open-loop conformations to a
greater degree than the WT (Fig. 6B). Although the crystal
structure of Sph shows a more closed conformation of the loop,
the MD simulations indicate that this region is quite flexible in
solution, and that the distinction between open and closed
conformations is not so apparent, likely providing a scaffold with
balanced R-stereoselectivity and catalyst turnover.
tions; in nearly all cases, the favored enantiomer is predicted. In
agreement with the crystallographic and MD results reported
above, the erS/R values calculated when the loop is open show a
poorer correlation with the experimental values (SI Appendix, Fig.
S22). We examined how individual mutations alter the energetics
of the diastereomeric active sites and modulate the enantiose-
lectivity of the enzyme.
In the WT enzyme (Fig. 9 A and B), the small binding pocket
is sterically hindered by the phenol group of Tyr190, disfavoring
2
the orientation of the S atom (larger than CH ) in this region of
the active site. In addition, the same phenol ring engages in a
CH/π interaction with the β-methylene group (Fig. 9B). As a
‡
result, (R)-2 is slightly favored (calc. erS/R = 0.8). On the other
hand, the smaller O atom fits better in the small pocket, in-
creasing the contribution of the pro-S pathway for 3-oxacyclo-
pentanone (calc. erS/R ∼ 1) (Fig. 9A). Of note, the primary
catalytic contacts—namely, the hydrogen bonds between the
incipient alkoxide (COsubstrate) and Ser143/Tyr156—are perfectly
maintained throughout the entire trajectories for the four sys-
tems, a finding frequently observed in other mutants as well. The
subtle differences between the pro-R and pro-S pathways in favor
Subsequent to our MD simulations of the Michaelis–Menten
complexes, and to assess the ability of the active site to stabilize the
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Fig. 5. (A) Overlay of the crystal structures obtained for apo WT (red), NADP -bound WT (magenta), A94F (blue), E145S (green), and Sph (yellow). The
2
+
cofactor is shown as pink sticks; the Mg ions, as orange spheres. Mutations introduced in the studied variants are shown as cyan circles; the active sites, as
purple ellipses. (B) The substrate/cofactor binding region showing the open and closed conformations of the loop. (C–F) The Tyr190-Glu/SerS145-Asp150 triad
in different variants, showing the water and/or hydrogen bond network.
of the latter arise from the contacts between the ribose hydroxyl
OHsugar) and Tyr156/Lys160. An unexpected and very persistent
interaction between COsubstrate and OHsugar was also observed
and OHTyr190, is observed. These geometric features are shown
in SI Appendix, Figs. S24 and S29.
The E145S single mutation (Fig. 9 E and F) modifies the
complex hydrogen bond network at the active site, disrupting the
water-mediated (Fig. 5E) or direct interaction between Tyr190
and Glu145 and producing a new Tyr190–Asp150 interaction.
(
(
SI Appendix, Figs. S23 and S28).
In the A94F mutant (Fig. 9C), forcing the 3-thiacyclopenta-
none ring to adopt a reactive pro-S orientation in the presence of
the bulky Phe94 in the large pocket induces a conformational
change in Tyr190, which no longer binds to E145 and occasionally
binds to the nicotinamide moiety. In this way, the formerly small
binding pocket is significantly expanded to accommodate the
larger S atom, and the primary catalytic contacts (COsubstrate with
Ser143/Tyr156) are maintained very close to the optimal values,
increasing S-enantioselectivity (SI Appendix, Figs. S24 and S29).
The same effect is produced by the Y190F mutation, in which
deletion of the OHTyr190 group disrupts its hydrogen bond network
with Glu145, Tyr249, and Asp150 and opens up the small pocket
to accommodate the endocyclic S and thus promote S-selectivity
(
Fig. 9D and SI Appendix, Figs. S26 and S31).
The hydrophobic cage formed by Y190, Tyr156, and especially
Phe94 (Figs. 7 and 9C) interacts strongly with the 3-oxacyclo-
pentanone ring through CH/π contacts in the pro-R TS complex.
Consequently, the catalytic Ser143 is displaced from its optimal
position and replaced by the OHsugar group, revealing the in-
ability of this enzyme to stabilize this particular TS. This obser-
vation agrees with the fivefold decrease in activity of this mutant
with respect to the WT and the enhancement of S-selectivity. In
Fig. 6. (A) Overlay of 10 snapshots derived from MD simulations of WT
KRED in complex with 2. The substrate-binding loop is highlighted for the
open (green), intermediate (purple), and closed (blue) conformations. One
representative binding pose of the cofactor is shown as sticks, and the active
site is shown as a yellow ellipse. The Cα of residues 93–96, 195, and 199 are
shown as spheres, and the distances analyzed to define the conformation of
the loop are shown as yellow arrows. (B) Experimental relative activation
‡
fact, the pro-S TS complex (S)-1 is able to accommodate the
‡
barriers leading to the S and R alcohols (ΔΔG S-R) from cyclopentanones 1
smaller endocyclic O atom in the encumbered small pocket
without producing any conformational change in Tyr190, pre-
serving all of the catalytic interactions during the simulation. In
addition, an occasional hydrogen bond between the endocyclic O
and 2, versus the accumulated population of the substrate-binding loop in
the closed conformation (Cα–Cα distance between L195 and A/F94 <10 Å)
derived from multiple 100-ns MD trajectories of different KRED variants in
complex with substrates 1 and 2 in different orientations.
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‡
group of the substrate and the phenyl ring of Tyr190 in (R)-2
Fig. 9H) may be one source of the increased R-enantiose-
(
lectivity with 3-thiacyclopentanone, given that the catalytic con-
tacts are very well maintained in both approximations (SI
Appendix, Figs. S27 and S32).
Conclusion
Enzyme design and engineering for enantioselective synthesis
pose significant challenges for chemical biology that are re-
ceiving increasing attention (20). The highly enantioselective
enzymes obtained through DE for the reduction of 3-oxacyclo-
pentanone and 3-thiacyclopentanone are less active than the
WT, however. Our computational studies of the origins of ster-
eoselectivity in these evolved ketoreductases have revealed that
the induction of asymmetry can be achieved by blocking certain
reaction pathways rather than promoting others, yielding slower
but more selective processes. Through crystallographic and MD
simulations of selected variants, we have found that certain
mutations can regulate the conformation and flexibility of the
substrate-binding loop that can be transmitted to the active site
with various stereochemical consequences depending on the
substrate used in the reduction reaction.
Certain point mutations in the active site, such as A94F and
E145S, can invert the enantioselectivity for 3-thiacyclopentanone
reduction by further closing or opening of the substrate-binding
loop and reconfiguring the space in the binding pockets. A94F
generates a hydrophobic cage that traps both the reactant and TS
in unproductive binding modes or enlarges the small binding
pocket, whereas E145S shrinks the small binding pocket and
amplifies the difference in size between an S atom and a CH2
group, making it more discriminating. These results have major
implications for the computational design of new biocatalysts
and demonstrate the importance of including appropriate protein
conformational sampling in parallel with primary sequence design.
Fig. 7. (A and B) An unproductive binding pose of 3-thiacyclopentanone 2 in
A94F single mutant in two different orientations showing the hydrophobic cage
surrounding the substrate (A) and the perpendicular orientation of 2 with re-
spect to NADPH despite the conserved catalytic contacts (B). (C) Traces of se-
lected interactions monitored along the MD trajectory: Cα–Cα distance between
Leu195 and Phe94 (loop conformation, in purple) and O–O distances between
the ketone carbonyl and the catalytic Ser143 (in blue) and Tyr156 (in green).
This new contact shrinks the small binding pocket and precludes
direct interaction of the OHTyr190 group with the endocyclic O of
3
-oxacyclopentanone in the pro-S orientation (SI Appendix, Fig.
Materials and Methods
S30). This reactive pose is also very much disfavored with
Generation and Screening of the Mutant Library. Experiments were run fol-
lowing the Codexis standard KRED panel screening protocol (SI Appendix,
Materials and Methods). A total of 175 KRED variants, including L. kefir
KRED, with 1–16 mutations from L. kefir WT KRED, were mixed with each
substrate, 1 and 2, in 96-well plates. Each well contained one substrate, cell
3
-thiacyclopentanone, in which the larger S atom clashes with the
aromatic sidechain of Tyr190 (Fig. 9E). The poorer binding
properties of this variant are reflected by the departure of the TS
‡
complex from the active site observed in (S)-2 after ∼40 ns,
+
which is the cause of the exceedingly high calculated R-enan-
tioselectivity (SI Appendix, Figs. S25 and S30). In contrast, both
substrates fit better in the pro-R orientation (Fig. 9F), in which
the primary catalytic contacts stabilizing the incipient alkoxide
are very well maintained, in agreement with the increased se-
lectivity observed experimentally for 2. Again, the major source
of the increased stereoselectivity in this mutant—which retains
one-half the activity of the WT—seems to be the destabilization
of one stereoselective pathway (pro-S in this case), with the other
maintained or slightly stabilized.
lysate, which included one KRED variant, NADP sodium salt, and trietha-
nolamine in isopropanol. Reactions were allowed to proceed at room tem-
perature for 24 h. Gas chromatography was used to determine the
conversion and the er of the resulting alcohol.
Crystallization Experiments. Crystals of the WT KRED and variants were grown
using a hanging-drop vapor diffusion method. Crystals of the WT KRED, both
The final Sph mutant (Fig. 9 G and H) is the most R-selective
variant for 2 (experimental erS/R = 0.004), but our MD protocol
largely underestimates this selectivity (calc. erS/R = 0.6). On the
other hand, this variant is also the less S-selective one for 1
(experimental erS/R = 1.3), but in this case our method overes-
timates the selectivity (calc. erS/R = 27). These results show the
limitations of the current protocol for highly evolved mutants,
for which additional factors related to protein stability and cat-
alyst turnover may play important roles in enantioselective
catalysis.
Similar geometric features to those observed in the E145S
mutant were observed in these systems, namely the formation of
a persistent Tyr190–Asp150 interaction, although the contrac-
tion of the small pocket is less apparent. In fact, in the four
analyzed systems, the cyclopentanone moieties of the TS com-
plexes seem to fit in the active site without significant steric
hindrance. Enhanced CH/π interactions between the β-CH2
Fig. 8. (A) Catalytic distances (a–d) monitored in the MD simulations with
the theozymes. (B) Logarithmic graph of the experimental vs. computed erS/R
derived from the population of the catalytically competent poses in the MD
trajectories [calc. erS/R = %(S)/%(R)]. The values for 3-oxacyclopentanone and
3-thiacyclopentanone are shown in red and blue, respectively.
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‡
Fig. 9. Overlays of 12 snapshots obtained along 160-ns MD simulations with TS complexes of selected KRED variants. (A) WT and (S)-1 . (B) WT and (R)-2 .
‡
‡
‡
‡
‡
‡
(
C) A94F and (S)-2 . (D) Y190F and (S)-2 . (E) E145S and (S)-2 . (F) E145S and (R)-2 . (G) Sph and (S)-2 . (H) Sph and (R)-2 . The cofactor and substrate are shown in
pink and purple, respectively. The catalytic residues Ser143, Tyr156, and Lys160 are in blue; Tyr/Phe190 and Asp150 are in gray; and Leu195, Ala/Phe/Ser94, and
Glu/Ser145 are in light green. The approximate extension of the active site is shown as a yellow ellipse. The stability of these TS complexes according to the
preservation of the catalytic contacts throughout the MD trajectories is indicated as favored (✓) or disfavored (X).
+
apo- and NADP -bound, were grown at room temperature. Crystals of A94F
and E145S were grown at 4 °C. Buffer compositions, concentrations, and
other details are provided in SI Appendix, Materials and Methods.
tries using the M06-2X method (24) and 6–311+G(d,p) basis set, and with
CPCM in water. Further details of the methodology are provided in SI Ap-
pendix, Materials and Methods.
Data Collection and Structure Determination. X-ray diffraction data were
collected at the Advanced Photon Source (Argonne National Laboratory),
Beamline NECAT 24ID-C, using a DECTRIS PILATUS-6M pixel detector. Data
reduction and scaling were performed using XDS for all crystals. Diffraction to
MD Simulations. Crystallographic structures of the tetrameric proteins served
as starting geometries. Parameters for the cofactor, substrates, and TS
complexes were generated with the antechamber module of Amber 12 (25)
using the general Amber force field (GAFF), and atomic partial charges were
derived from QM electrostatic potentials. Topology and initial coordinates
are available in Amber format in Datasets S1–S8. Each protein complex was
immersed in a truncated octahedral box with a 10-Å buffer of TIP3P (26)
2
.2-Å resolution was observed for the WT KRED, diffraction to 1.6-Å reso-
lution was observed for A94F, and diffraction to 1.7-Å resolution was ob-
+
served for E145S. X-ray diffraction data for the NADP -bound WT KRED
crystal were collected at the University of California Los Angeles, using a
Rigaku FRE+ rotating anode and a Rigaku HTC image plate detector. Dif-
fraction to 2.1-Å resolution was observed. All of the crystal structures were
determined by the molecular replacement method using the PHASER pro-
gram and using the KRED from L. brevis (Protein Data Bank ID code 1NXQ) as
the search model. Specifics of the crystal preparation, data collection, and
refinement are reported in SI Appendix, Materials and Methods.
+
−
water molecules, and was neutralized by adding explicit counterions (Na Cl ). All
subsequent simulations were performed using the Stony Brook modification of
the Amber 99 force field (27). Equilibration and production procedures are de-
scribed in SI Appendix, Materials and Methods.
ACKNOWLEDGMENTS. We thank M. Collazo for help with protein crystal-
lization and M. Sawaya for helpful discussions. We also thank M. Capel,
K. Rajashankar, N. Sukumar, F. Murphy, and I. Kourinov of Northeastern
Collaborative Access Team Beamline ID-24 at the Advanced Photon Source
of Argonne National Laboratory, supported by National Institutes of Health
Grants P41 RR015301 and P41 GM103403. Use of the Advanced Photon
Source is supported by the US Department of Energy under Contract DE-
AC02-06CH11357. This work was performed using the Extreme Science and
Engineering Discovery Environment, which is supported by the National
Science Foundation (Grant OCI-1053575) and the University of California, Los
Angeles (UCLA) Institute for Digital Research and Education’s Hoffman2
computer cluster. The UCLA X-Ray Core Facility is supported in part by US
Department of Energy Grant DE-FC03-02ER63421. This research was sup-
ported in part by the National Institutes of Health, National Institute of
General Medical Sciences (Grants GM036700, GM097200, and GM075962).
S.O. is supported by the Ministerio de Economía y Competitividad (Juan de la
Cierva Postdoctoral Grant JCI-2012-14438) and the European Commission
Career Integration Grants program (Grant PCIG14-GA-2013-630978). C.M.B.
was supported by NIH, NIGMS Training Grant T32 GM067555-11.
M
Kinetics Experiments. To measure the efficiency (kcat/K ), the concentration
of enzyme was fixed at 0.05 mM, and the concentration was varied from 10
to 160 mM for 1 and from 0.2 to 1 mM for 2, because catalysis of its re-
duction is less efficient. DMSO was added to a final concentration of
2
% (vol/vol) to facilitate solubilization of the substrates. The reactions took
place in 150 mM phosphate buffer (pH 7.0) at 25 °C. The final reaction so-
lution was 7% (vol/vol) glycerol with 2 mM MgSO and 1 mM NADPH. The
4
rate of NADPH depletion was measured using a Tecan Infinite M200 Pro
plate reader by recording the absorbance of the reaction solutions at 340 nm
relative to an NADPH standard.
QM Calculations. All calculations were performed with Gaussian 09 (21). The
geometries were optimized with the TPSS functional (22) and 6–31G(d) basis
set with the conductor-like polarizable continuum model (CPCM) (23) for
water. Single-point energy calculations were performed on these geome-
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