pubs.acs.

org/joc

Highly Enantioselective Mutant Carbonyl Reductases Created via

Structure-Based Site-Saturation Mutagenesis
Hongmei Li,‡ Yan Yang,‡ Dunming Zhu,*,†,‡ Ling Hua,‡,§ and
Katherine Kantardjieff^
†
State Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology,
Chinese Academy of Sciences, Tianjin 300308, China, ‡Department of Chemistry, Southern Methodist
University, Dallas, Texas 75275, United States, §China Research Center, Genencor, A Danisco Division,
Shanghai 200335, China, and ^Department of Chemistry, California State Polytechnic University, Pomona,
California 91768, United States

zhu_dm@tib.cas.cn
Received August 6, 2010

A carbonyl reductase from Sporobolomyces salmonicolor reduced para-substituted acetophenones with low
enantioselectivity. Enzyme-substrate docking studies revealed that residues M242 and Q245 were in close
proximity to the para-substituent of acetophenones in the substrate binding site. Site-saturation mutagen-
esis of M242 or Q245, and double mutation of M242 and Q245 were performed in order to enhance the
enzyme’s enantioselectivity toward the reduction of para-substituted acetophenones. Three Q245 mutants
were obtained, which inverted the enantiopreference of product alcohols from (R)- to (S)-configuration
with high ee values (Org. Lett. 2008, 10, 525-528). Four M242 mutant enzymes also showed greater
preference for the formation of (S)-enantiomeric alcohols than the wild-type enzyme, but to a much less
extent than Q245 mutants. M242/Q245 double variations not only greatly affect the enantiomeric purity of
the product alcohols, but also invert the enantiopreference, demonstrating that these residues play a critical
role in determining the enantioselectivity of these ketone reductions. The kinetic parameters of these mutant
enzymes indicated that residues 242 and 245 also exert an effect on the catalytic activity of this carbonyl
reductase. Highly enantioselective mutant carbonyl reductases were created by site-saturation mutagenesis,
among which the one bearing double mutations, M242L/Q245P, showed the highest enantioselectivity that
catalyzed the reduction of the tested para-substituted acetophenones to give (S)-enantiomeric products in
g99% ee with only one exception of p-fluoroacetophenone (92% ee).

Introduction other approaches.1-5 The information about the substrate

specificity and enantioselectivity of enzymes is very valuable
Enantiomerically pure chiral alcohols are important inter-
mediates in the fine chemical and pharmaceutical industries.
In recent years, biocatalytic reduction of ketones has in- (1) Moore, J. C.; Pollard, D. J.; Kosjek, B.; Devine, P. N. Acc. Chem. Res.
2007, 40, 1412.
creasingly grown as a general method of choice for the (2) De Wildeman, S. M. A.; Sonke, T.; Schoemaker, H. E.; May, O. Acc.
synthesis of chiral alcohols and efficiently complements Chem. Res. 2007, 40, 1260.
(3) Kroutil, W.; Mang, H.; Edegger, K.; Faber, K. Curr. Opin. Chem.
Biol. 2004, 8, 120.
*To whom correspondence should be addressed. Tel: (þ86)-22-84861962. (4) Huisman, G. W.; Liang, J.; Krebber, A. Curr. Opin. Chem. Biol. 2010,
Fax: (þ86)-22-84861996. 14, 122.

DOI: 10.1021/jo101541n Published on Web 10/22/2010 J. Org. Chem. 2010, 75, 7559–7564 7559
r 2010 American Chemical Society
JOC Article Li et al.

and instructive in guiding the selection of appropriate bio- SCHEME 1. Enzymatic Reduction of Para-Substituted Aceto-
catalysts for a specific transformation.6-9 Correlating the phenones Catalyzed by SSCR Mutant Enzymes with a Cofactor
substrate profile of an enzyme with its sequential and Regeneration System of D-Glucose Dehydrogenase and D-Glucose
structural information not only provides valuable insight
into the understanding of how enzymes control activity and
enantioselectivity but also facilitates our efforts to (semi)
rationally design novel enzymes with desired substrate spec-
ificity and enantioselectivity.10-13 Holding these in mind, we
have studied substrate specificity and enantioselectivity of
several carbonyl reductases from different origins that cat-
alyze reduction of ketones of diverse structures. To our
delight, these carbonyl reductases are capable of converting
aryl/alkyl ketones and R-/β-ketoesters to the corresponding M242 and Q245 are in close proximity to the para-substit-
chiral alcohols with excellent enantiomeric purity.14-20 In uent of the acetophenones with hydrogen bonding or hydro-
the course of our studies on a carbonyl reductase from phobic interactions.25,26 These simulation results are quali-
Sporobolomyces salmonicolor (SSCR), it has been found that tatively consistent with the observed low enantio selectivity
SSCR showed an unusually broad substrate range including in the SSCR-catalyzed reduction of para-substituted acet-
aliphatic, aromatic ketones, R- and β-ketoesters, and steri- ophenones, and such close interaction was conjectured to
cally bulky aryl alkyl ketones and diaryl ketones.15,16,20 This have played significant roles in determining the enzyme’s
enzyme showed low enantioselectivity for the reduction of enantioselectivity. Therefore, the residues M242 and Q245 in
para-substituted acetophenones (14-59% ee), although it the catalytic site were identified as the mutation targets to
catalyzed the reduction of other ketones to the corre- improve the enzyme enantioselectivity. Single and double
sponding chiral alcohols with excellent enantiomeric saturation mutagenesis of residues M242 and Q245 were
purity.15,16 Recently, the X-ray structures of this carbonyl performed, and the resulting mutant libraries were screened
reductase and its complex with a coenzyme, NADPH, have for enhanced enantioselectivity toward the reduction of
been determined.21 A substrate-enzyme docking study of para-substituted acetophenones. The preliminary results of
p-methoxyacetophenone into the crystal structure of SSCR site-saturation mutagenesis of residue Q245 were previously
was performed with ICM-Pro 3.4.9d22-24 in order to better communicated,26 herein a detailed account of these studies
understand the enantioselective versatility in this ketone are presented.
reduction. During these simulations, two opposite orienta-
tions of p-methoxyacetophenone, which yield (S)-alcohol Results and Discussion
isomer or the (R)-counterpart, respectively, have been found
Focused libraries of mutants were created by means of
to be energetically close to each other in the high-scoring
site-saturation mutagenesis at residues M242 and Q245 in
docking conformations. In both conformations, residues
the catalytic cavity of the carbonyl reductase from S. salmo-
(5) Martı́n-Matute, B.; Edin, M.; Bog
ar, K.; Kaynak, F. B.; B€ ackvall, nicolor. The resulting mutant libraries were screened with
J.-E. J. Am. Chem. Soc. 2005, 127, 8817. p-methoxyacetophenone as substrate to select colonies
(6) Burton, S. G.; Cowan, D. A.; Woodley, J. M. Nat. Biotechnol. 2002, which showed close or higher activity than the wild-type
20, 37.
(7) Ema, T.; Yagasaki, H.; Okita, N.; Nishikawa, K.; Korenaga, T.; SSCR enzyme. The enantioselectivity of these selected co-
Sakai, T. Tetrahedron: Asymmetry 2005, 16, 1075. lonies was then measured by chiral gas chromatography. The
(8) Inoue, K.; Makino, Y.; Itoh, N. Tetrahedron: Asymmetry 2005, 16,
2539.
preliminary results obtained from the screening of the site-
(9) Kaluzna, I. A.; Matsuda, T.; Sewell, A. K.; Stewart, J. D. J. Am. saturation mutagenesis library at residue Q245 were pre-
Chem. Soc. 2004, 126, 12827. viously reported in a communication26 and are not repeated
(10) Phillips, R. S. Can. J. Chem. 2002, 80, 680.
(11) Reetz, M. T.; Carballeira, J. D.; Peyralans, J.; Hobenreich, H.; here. In screening the saturation mutagenesis library of
Maichele, A.; Vogel, A. Chem.;Eur. J. 2006, 12, 6031. residue M242, four mutants were selected with higher
(12) Paradisi, F.; Conway, P. A.; Maguire, A. R.; Engel, P. C. Org. activity than others in the NADPH-adjuvant reduction of
Biomol. Chem. 2008, 6, 3611.
(13) Babtie, A.; Tokuriki, N.; Hollfelder, F. Curr. Opin. Chem. Biol. 2010, p-methoxyacetophenone. Among these mutant enzymes,
14, 200. M242Y, M242C, and M242G maintained the same stereo
(14) Zhu, D.; Yang, Y.; Hua, L. J. Org. Chem. 2006, 71, 4202.
(15) Zhu, D.; Yang, Y.; Buynak, J. D.; Hua, L. Org. Biomol. Chem. 2006,
preference (R) with the wild-type SSCR but they catalyzed
4, 2690. the reduction with lower enantioselectivity than the latter.
(16) Zhu, D.; Hua, L. J. Org. Chem. 2006, 71, 9484. However, M242D produced (S)-1-(p-methoxyphenyl)ethanol
(17) Zhu, D.; Malik, H. T.; Hua, L. Tetrahedron: Asymmetry 2006, 17,
3010. with ee values of 39%.
(18) Yang, Y.; Zhu, D.; Piegat, T. J.; Hua, L. Tetrahedron: Asymmetry These mutant SSCR enzymes were further studied to
2007, 18, 1799. determine their enantioselectivity toward the reduction of
(19) Zhu, D.; Stearns, J. E.; Ramirez, M.; Hua, L. Tetrahedron 2006, 62,
4535. other para-substituted acetophenones (Scheme 1). The co-
(20) Li, H.; Zhu, D.; Hua, L.; Biehl, E. R. Adv. Synth. Catal. 2009, 351, factor NADPH was regenerated with D-glucose dehydro-
583.
(21) Kamitori, S.; Iguchi, A.; Ohtaki, A.; Yamada, M.; Kita, K. J. Mol.
genase and D-glucose. The results are summarized in Table 1.
Biol. 2005, 352, 551. It can be seen that when compared to the wild-type SSCR,
(22) Schapira, M.; Abagyan, R.; Totrov, M. J. Med. Chem. 2003, 46,
3045.
(23) Schapira, M.; Raaka, B. M.; Das, S.; Fan, L.; Totrov, M.; Zhou, Z.; (25) Cundari, T. R.; Dinescu, A.; Zhu, D.; Hua, L. J. Mol. Model. 2007,
Wilson, S. R.; Abagyan, R.; Samuels, H. H. Proc. Natl. Acad. Sci. U.S.A. 13, 685.
2003, 100, 7354. (26) Zhu, D.; Yang, Y.; Majkowicz, S.; Pan, T. H.-Y.; Kantardjieff, K.;
(24) An, J.; Totrov, M.; Abagyan, R. Mol. Cell. Proteomics 2005, 4, 752. Hua, L. Org. Lett. 2008, 10, 525.

7560 J. Org. Chem. Vol. 75, No. 22, 2010

Li et al.
JOC Article
TABLE 1. Reduction of Para-Substituted Acetophenones with M242 Mutant Enzymes of SSCRa
substrate SSCR-WT ee (%) M242Y ee (%) M242D ee (%) M242C ee (%) M242G ee (%)
p-H 42 (R) 12 (S) 82 (S) 13 (S) 54 (S)
p-F 46 (R) 41 (S) 90 (S) 54 (S) 70 (S)
p-Cl 14 (R) 36 (S) 77 (S) 27 (S) 62 (S)
p-Br 42 (R) 22 (S) 61 (S) 4 (R) 52 (S)
p-CH3 59 (R) 21(R) 43 (S) 38 (R) 4 (S)
p-OCH3 57 (R) 7 (R) 39 (S) 18 (R) 6 (R)
p-C(CH3)3 31 (R) 93 (S) >99 (S) 96 (S) 90 (S)
p-CF3 17 (S) 28 (S) 37 (S) 5 (S) 17 (S)
a
The ee values were determined by chiral GC analysis.27

TABLE 2. Reduction of Para-Substituted Acetophenones with M242/Q245 Double Mutant Enzymes of SSCRa
substrate SSCR-WT ee (%) M242L/Q245P ee (%) M242F/Q245T ee (%) M242C/Q245L ee (%) M242L/Q245T ee (%)
p-H 42 (R) 74 (S) 42 (S) 17 (S) 58 (S)
p-F 46 (R) 92 (S) 90 (S) 16 (S) 94 (S)
p-Cl 14 (R) 99 (S) 94 (S) 50(S) 98 (S)
p-Br 42 (R) >99 (S) 89 (S) 24 (S) 99 (S)
p-CH3 59 (R) 99 (S) 72 (S) 20 (S) 95 (S)
p-OCH3 57 (R) 99 (S) 92 (S) 36 (S) 97 (S)
p-C(CH3)3 31 (R) >99 (S) 95 (S) 99 (S) 99 (S)
p-CF3 17 (S) >99 (S) 98 (S) 94 (S) 99 (S)
a
The ee values were determined by chiral GC analysis.27

which catalyzed the reduction to give (R)-enantiomer, these The library was screened with p-methoxyacetophenone as
mutant enzymes (M242Y, M242D, M242C, and M242G) the substrate for higher activity than the wild-type enzyme,
disfavored the formation of (R)-configurated chiral alcohols (S)-1-(p-Methoxyphenyl)ethanol was the major enantiomer of
for reductions of all para-substituted acetophenones to such the product alcohol for all four mutant enzymes in which three of
an extent that the (S)-enantiomer of product alcohols was them exhibited high enantioselectivity with greater than 92% ee.
obtained in most cases. Especially for p-tert-butylacetophe- Some other para-substituted acetophenones were also
none, these mutant SSCR enzymes exhibited inverted en- studied with these double mutant enzymes to examine their
antiopreference and enhanced enantioselectivity. It is clear enantioselectivity (Table 2). As shown in Table 2, all four
that the residue 242 in the catalytic cavity plays an important double mutant enzymes greatly increased the (S)-enantio-
role in determining the enantioselectivity for the reduction of meric preference to an extent that the absolute configuration
the para-substituted acetophenones, but to a less extent than of major products was inverted from (R) to (S). Among
residue Q245. Among the four mutants, M242D exerts the them, mutant M242L/Q245P possessed the highest enantio-
greatest effect on the enzyme enantioselectivity. If the sub- selectivity in the reduction of these ketones, which was higher
stituent amino acids are lined up according to their hydro- than those of the mutant Q245P, suggesting that replace-
phobicity as M > C, Y, G . D, it is intriguing to observe that ments of M242 with L and Q245 with P had a synergic effect
the replacement of hydrophobic methionine with polar on preferable formation of (S)-enantiomeric products. Com-
amino acids cysteine, tyrosine, and glycine has altered the pared to Q245L, mutant M242C/Q245L showed much lower
enzyme spatial preference toward the S configuration. This enantioselectivity in most cases. The preference for the (S)-
tendency is first reflected on the decrease in the number of enantiomer formation of these related mutant enzymes
substrates with R preference (SSCR-WT = 7, M242C = 3, followed the order wild-type < M242C < M242C/Q245L
M242Y = 2, M242G = 1 and M242D = 0). Second, this <Q245L, indicating that M242C had a negative effect on the
hydrophobicity effect was also reflected in enantiomeric (S)-enantiomeric preference of Q245L. In contrast to this,
values, e.g., for p-methoxyacetophenone, the ee value toward M242C had a positive effect on the (S)-enantiomeric pre-
the R enantiomer dropped from 57% (the wild-type) to 6% ference of wild-type enzyme as described above. Mutant
(M242G). The replacement with negative charged aspartic M242L/Q245T exhibited higher enantioselectivity than
acid then led S isomer alcohols as predominant products for mutant M242F/Q245T. The synthetic application of these
all eight substrates. For p-trifluoromethylacetophenone, the mutant enzymes was demonstrated by the reduction of
major product was (S)-enantiomer with wild-type and mu- p-chloroacetophenone with mutant M242L/Q245P at prepara-
tant enzymes as biocatalyst; mutation of residue M242 did tive scale, giving (S)-1-(p-chlorophenyl)ethanol in 92% yield.
not greatly affect the enzyme enantioselectivity. The kinetic parameters of these mutant carbonyl reduc-
It has been demonstrated that mutation of either residue tases were measured and their overall catalytic efficiencies
Q245 or M242 greatly tuned the enantioselectivity of car- (kcat/Km) toward these para-substituted acetophenones are
bonyl reductase SSCR toward para-substituted acetophe- presented in Figures 1-3. From Figure 1, it can be seen that
nones. A question has arisen as to whether a highly enantio- the overall catalytic efficiencies of mutant carbonyl reduc-
selective mutant carbonyl reductase could be created by tases M242Y, M242D, and M242G were lower than wild-
double mutation of residues M242 and Q245? With interest type enzymes, while for mutant M242C, the kcat/Km values
in searching for novel highly enantioselective carbonyl re- were comparable with those of the wild-type enzyme for
ductases, we constructed a focused double mutant library by most substrates with the exceptions of p-chloro (twice
site-saturation mutagenesis at both residues 242 and 245. higher) and the unsubstituted acetophenone (half high).
J. Org. Chem. Vol. 75, No. 22, 2010 7561
JOC Article Li et al.

FIGURE 1. The catalytic efficiency (kcat/Km, min-1 3 mM-1) of

SSCR wild-type and M242 mutant enzymes toward reduction FIGURE 3. The catalytic efficiency (kcat/Km, min-1 3 mM-1) of
of acetophenones: (1) unsubstituted, (2) p-fluoro, (3) p-chloro, (4) SSCR wild-type and M242/Q245 double mutant enzymes for the
p-bromo, (5) p-methyl, (6) p-methoxy, (7) p-tert-butyl, and (8) reduction of acetophenones: (1) unsubstituted, (2) p-fluoro, (3)
p-trifluoromethyl. p-chloro, (4) p-bromo, (5) p-methyl, (6) p-methoxy, (7) p-tert-butyl,
and (8) p-trifluoromethyl.

corresponding “anti-Prelog” (R)-alcohols. As a comparison,

the wild-type enzyme was not able to reduce these ketones
and provided (S)-alcohols as the major products for the
reduction of smaller ketones.28 Other examples are the
substrate-binding residue exchange of stereocomplementary
ketoreductase domains leading to the reversion of stereo-
chemistry. For example, mutation of residues in motifs I and
II in ketoreductase domains eryKR1 and eryKR2 from the
erythromycin polyketide synthase altered the stereochemical
outcome in reduction of (2R,S)-2-methyl-3-oxopentanoic
acid N-acetyl-cysteamine thioester.29,30 In the study to con-
firm the hypothetical tropinone binding mode, replacement
of five substrate-binding residues of one tropinone reductase
(TR-1) with those found in the corresponding positions of
the other tropinone reductase (TR-2) in the biosynthetic
FIGURE 2. The catalytic efficiency (kcat/Km, min-1 3 mM-1) of pathway of tropane alkaloids resulted in the switch of
SSCR wild-type and Q245 mutant enzymes toward reduction stereospecificity of TR-1 into that of TR-2 and vice versa.31
of acetophenones: (1) unsubstituted, (2) p-fluoro, (3) p-chloro, (4) These structure-based mutageneses revealed some insights
p-bromo, (5) p-methyl, (6) p-methoxy, (7) p-tert-butyl, and (8)
into how these enzymes control their stereoselectivity. The
p-trifluoromethyl.
scarcity of such study might be due to the difficulty of
The Q245 mutant and M242/Q245 double mutant en- implementing a high-throughput method to determine en-
zymes showed higher overall catalytic efficiencies than the antioselectivity in the ketone reduction that allows the rapid
wild-type enzyme, especially for the acetophenones with screening of a large number of mutants. The present study
electron-withdrawing groups such as chloro, bromo, and demonstrates that enzyme-substrate docking-guided site-
trifuoromethyl (Figures 2 and 3). These results suggest that mutagenesis is a practical approach to access highly enan-
residues 242 and 245 not only play an important role in tioselective carbonyl reductase enzymes.
determining the enzyme’s enantioselectivity, but also affect
the catalytic activity toward the reduction of para-substi- Conclusions
tuted acetophenones, and residue 245 exerts a much greater
Although a carbonyl reductase from Sporobolomyces
influence on both enantioselectivity and catalytic activity.
salmonicolor catalyzed the reduction of various ketones
Naturally occurring enzymes are not always highly active
with diverse structures to highly enantiomerically pure
and/or stereoselective for unnatural substrates. Improve-
products, it reduced para-substituted acetophenones with
ment in enzyme activity and enantioselectivity is often
low enatioselectivity. Enzyme-substrate docking studies
necessary to meet the synthetic needs. Up to now, reports
on the enantioselectivity engineering of carbonyl reductase
are still very limited. Recently, Philips’s group has reported (28) Musa, M. M.; Lott, N.; Laivenieks, M.; Watanabe, L.; Vieille, C.;
Phillips, R. S. ChemCatChem 2009, 1, 89.
that secondary ADH from Thermoanaerobacter ethanolicus (29) Baerga-Ortiz, A.; Popovic, B.; Siskos, A. P.; O’Hare, H. M.; Spiteller,
(TeSADH) bearing a single mutation I86A catalyzed the D.; Williams, M. G.; Campillo, N.; Spencer, J. B.; Leadlay, P. F. Chem. Biol.
reduction of benzylic and heteroaryl ketones to give the 2006, 13, 277.
(30) O0 Hare, H. M.; Baerga-Ortiz, A.; Popovic, B.; Spencer, J. B.;
Leadlay, P. F. Chem. Biol. 2006, 13, 287.
(27) Zhu, D.; Rios, B. E.; Rozzell, J. D.; Hua, L. Tetrahedron: Asymmetry (31) Nakajima, K.; Kato, H.; Oda, J. i.; Yamada, Y.; Hashimoto, T. J.
2005, 16, 1541. Biol. Chem. 1999, 274, 16563.

7562 J. Org. Chem. Vol. 75, No. 22, 2010

Li et al.
JOC Article
with p-methoxyacetophenone revealed that the observed low cultures were induced with isopropyl-β-D-thiogalactopyrano-
enantioselectivity was consistent with two energetically close side (IPTG, 0.1 mM), and incubated at 30 C for 6 h. The cells
conformations of substrate in the binding site. Residues M242 were harvested by centrifugation at 4100 rpm for 30 min and
and Q245 in the substrate binding site were identified as the key lysed with 400 U of Ready-Lyse lysozyme (Epicenter, Inc.) in
residues for enzyme engineering to create highly enantioselective 50 μL of TES buffer (100 mM NaCl, 1 mM EDTA, 10 mM Tris-
HCl, pH 7.5). The lysis reactions were incubated at room
carbonyl reductases for the reduction of para-substituted acet-
temperature for 30 min, and 300 μL of potassium phosphate
ophenones. Site-saturation mutagenesis of single point mutation buffer (100 mM, pH 6.5) was added to the mixture. The diluted
at M242 or Q245 and of double point mutation of these two cell-free extract thus obtained after centrifugation was used in
residues was performed. Four M242 variants, three Q245 var- the activity assays, which were carried out by spectrophotomet-
iants, and four M242/Q245 double mutant enzymes were ob- rically monitoring the disappearance of NADPH at 340 nm with
tained by screening of these focused mutant libraries. All these 40 -methoxyacetophenone as the substrate. The colonies, which
mutant enzymes showed stronger preference for the formation of showed activity close to or higher than the wild-type SSCR
(S)-configurated product alcohols in the reduction of para- enzyme, were cultivated in 15 mL of LB media containing 100
substituted acetophenones than the wild-type SSCR, leading μg/mL ampicillin at 37 C for 12 h. The cell-free lysates were
to the configuration invertion of the major product alcohols prepared as described above and used in the 1 mL scale reactions
to determine the enantioselectivity. The reaction mixtures con-
from (R) to (S) in most cases. Especially for the Q245 mutant
taining 300 μL of lysate, 0.5 mg of NADPH, 12.5 mM
and the double mutant enzymes, (S)-alcohols were obtained in a 40 -methoxyacetophenone, 0.5 mg of GDH, and 4.0 mg of
very high enantiomeric purity. Similarly, residues M242 and glucose in 1 mL of potassium phosphate buffer (100 mM, pH
Q245 also alter the enzyme activity, but M242 has less effect than 6.5) were shaken at room temperature for 12 h. The ee value
Q245, as shown in Figures 1-3. These studies indicated that of the product alcohol was measured by chiral GC analysis.
residues M242 and Q245 not only play a crucial role in determin- The plasmid DNAs from the clones, which showed some
ing the enantioselectivity of these ketone reductions, but also enantioselectivity, were isolated and sequenced. Confirmed
exert some effect on the enzyme’s catalytic activity. The struc- plasmid DNAs were transformed into E. coli competent cell
ture-based site-saturation mutagenesis created highly enantiose- Rosetta 2(DE3)pLysS.
lective mutant carbonyl reductases. Among them, double Screening of M242 and Q245 Double Mutant Library. Three
thousand colonies were picked up randomly from the double
mutant enzyme M242L/Q245P showed the highest enantioselec-
mutant library. A similar procedure described above was fol-
tivity, which catalyzed the reduction of the tested para-substi- lowed, using the wild type and mutant Q245P as references. The
tuted acetophenones to give (S)-enantiomeric products in plasmid DNAs from the clones, which showed higher enantios-
g99% ee, and the only exception was p-fluoroacetophenone electivity, were isolated and sequenced. Confirmed plasmid
with 92% ee. As such, the in silico docking guided semirational DNAs were transformed into E. coli competent cell Rosetta
approach should be a very valuable methodology for discovery 2(DE3)pLysS.
of new enzymes with synthetic advantages. Purification of SSCR Mutant Enzymes. SSCR mutants
were grown in LB medium containing 100 μg/mL ampicillin
and incubated at 37 C until the optical density reached 0.6 at
Experimental Section
600 nm. The expression was induced by 0.1 mM IPTG and the
M242 Mutant Library. The carbonyl reductase gene from cell culture was incubated at 30 C for another 6 h. The cells were
Sporobolomyces salmonicolor (SSCR) was cloned as described harvested by centrifugation at 4100 nm at 4 C for 30 min. The
before.15 The plasmid was used as the template for the Quick cell pellet was resuspended in potassium phosphate buffer (100
Change site-directed mutagenesis polymerase chain reaction mM, pH 7.4) and the cells were disrupted by a High Pressure
(PCR). The forward primer was 50 -TTCCCCGGCTCTGGC- Homogenizer. The cell-free extract was mixed with an equal
TCTGNNKCCACCGCAGT ACTACGTT-30 and the reverse volume of PEI solution (0.25% polyethyleneimine, NW 40-60
primer was 50 -AACGTAGTACTGCGGTGGMNNCAGAG- K, 6% NaCl, 100 mM Borax, pH 7.4) to remove lipids. The
CCAGAGCCGGGGAA-30 . PCR was carried out with pfx supernatant was precipitated with 55% ammonium sulfate. The
polymerase under the following conditions: the reaction was precipitate was resuspended in potassium phosphate buffer (10
started at 94 C (2 min), followed by 30 cycles: 94 C (30 s), 55 C mM, pH 7.4, 0.1 mM dithiothreitol), followed by diafiltration
(45 s), 68 C (6 min 48 s), with a final extension at 68 C (10 min). with potassium phosphate buffer (10 mM, pH 7.4, 0.1 mM
The reaction was carried out in 100 μL reaction volume contain- dithiothreitol) to remove the excess of ammonium sulfate. The
ing 0.5 pM of both primers, 210 ng genomic DNA, 1.0 mM resulting lysate was lyophilized to give the SSCR mutant
MgSO4, 0.2 mM dNTP, and 3.7 U of pfx polymerase. The PCR enzymes as white powders, which were used in the acetophenone
product was digested with DpnI restriction enzyme and elec- reduction to measure the activity and enantioselectivity.
tronically transformed to E. coli competent cell BL21(DE3). Kinetic Assay. The enzyme activity for ketone reduction was
M242 and Q245 Double Mutant Library. The double mutant determined by spectrophotometrically monitoring the reduc-
library construction followed the similar procedure described tion of NADPH at 340 nm at room temperature with a molar
above for M242. The forward primer was 50 -TTCCCCGG- extinction coefficient of 6220 M-1 cm-1. The reaction was
CTCTGGCTCTGNNKCCACCGNNKT-30 , and the reverse carried out in potassium phosphate buffer (100 mM, pH 7.0)
primer was 50 -AACAGCGGAAACGTAGTAMNNCGGTG- with 0.25 mM NADPH. The substrate concentration range was
GMNNCA-30 . between 0.1 and 2.4 mM. A microplate reader was applied for
Screening of M242 Mutant Library. Each of the 92 mutant the measurements and SigmaPlot version11 software was used
colonies along with 4 colonies expressing wild-type SSCR gene for calculation of the kinetic parameters.
were grown in 150 μL of Luria-Bertani (LB) medium supple- Measurement of Enantioselectivity. The enantioselectivity
mented with 100 μg/mL ampicillin at 37 C for 12 h in a 96-well of the enzymatic reduction of ketones was studied by using
microplate. For each colony, 4 μL of the overnight culture an NADPH recycle system. The general procedure was as
was diluted into 2 mL of LB medium containing 100 μg/mL follows: D-glucose (4 mg), D-glucose dehydrogenase (0.5 mg),
ampicillin and the culture was incubated at 37 C with shaking at NADPH (0.5 mg), the carbonyl reductase (SSCR, 0.5 mg), and
150 rpm until the optical density reached 1.2 (2 h). The cell ketone solution in DMSO (50 μL, 0.25 M) were mixed in a

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JOC Article Li et al.

potassium phosphate buffer (1 mL, 100 mM, pH 6.5) and the reaction mixture was shaken at room temperature for 24 h. The
mixture was shaken overnight at room temperature. The mix- reduction was completed as shown by GC analysis. The mixture
ture was extracted with methyl tert-butyl ether (1 mL). The was saturated with sodium chloride and extracted with methyl
organic extract was dried over anhydrous sodium sulfate and tert-butyl ether (3  30 mL). The organic extract was dried over
was subjected to chiral GC analysis to determine the enantio- anhydrous sodium sulfate and removal of solvent gave (S)-
meric excess. The absolute configurations of product alcohols 1-(p-chlorophenyl)ethanol14 (71.1 mg, yield 92%).
were identified by comparing the chiral GC data with the
standard samples.27
Acknowledgment. D.Z. thanks the Chinese Academy of
Enzymatic Reduction of p-Chloroacetophenone (Preparative
Scale). D-Glucose (20g L-1), D-glucose dehydrogenase (2 g L-1), Sciences for support from the Knowledge Innovation Program
NADPH (1 g L-1), and mutant M242L/Q245P enzyme (2 g L-1) (Grant No. KSCX2-YWG-031), Tianjin Municipal Science &
were dissolved in potassium phosphate buffer (100 mM, Technology Commission (No. 09ZCKFSH01000), and Minis-
pH 7.0), and 38 mL of the resulting solution was mixed with try of Science and Technology of China from the National Key
2 mL of p-chloroacetophenone solution in DMSO (0.25 M). The Technology R&D Program (No. 2008BAI63B07).

7564 J. Org. Chem. Vol. 75, No. 22, 2010