Microbial Baeyer-Villiger oxidation: Stereopreference and substrate acceptance of cyclohexanone monooxygenase mutants prepared by directed evolution

Article abstract of DOI:10.1021/ol0601040

An array of random mutants of cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871 was screened against a library of structurally diverse ketones for modifications in the substrate acceptance profile and stereopreference of the enzymatic B

Full text of DOI:10.1021/ol0601040

ORGANIC
LETTERS
2006
Vol. 8, No. 6
1221-1224
Microbial Baeyer−Villiger Oxidation:
Stereopreference and Substrate
Acceptance of Cyclohexanone
Monooxygenase Mutants Prepared by
Directed Evolution
Marko D. Mihovilovic,*,† Florian Rudroff,† Alexander Winninger,† Toni Schneider,‡
Frank Schulz,‡ and Manfred T. Reetz‡
Vienna UniVersity of Technology, Institute of Applied Synthetic Chemistry,
Getreidemarkt 9/163-OC, A-1060 Vienna, Austria, and Max-Planck-Institut fu¨r
Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mu¨lheim/Ruhr, Germany
mmihoVil@pop.tuwien.ac.at
Received January 13, 2006
ABSTRACT
An array of random mutants of cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871 was screened against a library
of structurally diverse ketones for modifications in the substrate acceptance profile and stereopreference of the enzymatic Baeyer Villiger
−
biooxidation. While the set of mutant biocatalysts was initially evolved for the enantiocomplementary oxidation of 4-hydroxycyclohexanone,
improved and/or divergent stereoselectivities were observed for several substrates. In addition, expanded substrate acceptance to facilitate
biotransformation of sterically demanding ketones was found.
The microbial Baeyer-Villiger oxidation (Scheme 1) has
received substantial attention in recent years as a valuable
ing during the past years, an increasing diversity of Baeyer-
Villiger monooxygenases (BVMOs) has become available
as versatile catalysts for such stereoselective transformations
which fulfill the requirements of sustainable and green
chemistry strategies.1 Until recently, widespread application
of such catalytic entities was limited by a general obstacle
of biocatalysis: While access to both antipodal forms of a
product can be achieved with de novo designed synthetic
catalysts, no generally applicable strategy is available to
provide enantiocomplementarity in biocatalysis.
Scheme 1. General Scheme of Baeyer-Villiger Oxidation
(1) (a) Mihovilovic, M. D.; Mu¨ller, B.; Stanetty, P. Eur. J. Org. Chem.
2002, 3711. (b) Kamerbeek, N. M.; Jannsen, D. B.; Berkel, W. J. H.; Fraaije,
M. W. AdV. Synth. Catal. 2003, 345, 667. (c) Mihovilovic, M. D.; Rudroff,
F.; Gro¨tzl, B. Curr. Org. Chem. 2004, 8, 1057. (d) Flitsch, S.; Grogan, G.
In Enzyme Catalysis in Organic Synthesis: A ComprehensiVe Handbook,
2nd ed.; Drauz, K., Waldmann, H., Eds.; VCH: Weinheim, 2002; Vol. 3,
p 1202. (e) Stewart, J. D. Curr. Org. Chem. 1998, 2, 195. (f) Roberts, S.
M.; Wan, P. W. H. J. Mol. Catal. B: Enzym. 1998, 4, 111. (g) Willetts, A.
Trends Biotechnol. 1997, 15, 55. (h) Walsh, C. T.; Chen, Y.-C. J. Angew.
Chem., Int. Ed. Engl. 1988, 27, 333.
tool for the transformation of prochiral or racemic ketones
into optically pure lactones which are attractive intermediates
in organic synthesis. Thanks to progress in genome decipher-
† Vienna University of Technology.
‡ Max-Planck-Institut fu¨r Kohlenforschung.
10.1021/ol0601040 CCC: $33.50
© 2006 American Chemical Society
Published on Web 02/21/2006

Two approaches to overcome this limitation were previ-
ously successfully implemented in the area of enzymatic
Baeyer-Villiger oxidation by our two groups. Taking
advantage of nature’s biodiversity, a platform of recombinant
E. coli strains overexpressing BVMOs of various microbial
origin was introduced2 with overlapping substrate profiles
for the conversion of various ketones. These catalysts display
enantiocomplementary stereospecificity3 and regiodivergent
biotransformations.4
Another strategy is based on the modification of a
particular biocatalyst of known characteristics. The combina-
tion of recent protocols from molecular biology5 with
efficient screening methods6 under a directing evolutionary
pressure (directed evolution) represents a powerful approach
toward obtaining new asymmetric catalysts.7 In the field of
enzymatic Baeyer-Villiger oxidation, a random strategy has
been successfully applied to modify the stereopreference of
cyclohexanone monooxygenase (CHMO) from Acinetobacter
sp. NCIMB 9871,8 and enantiodivergent biocatalysts were
evolved within two generations.9 In this process, a limited
number of amino acids become exchanged, and the genes
of the most promising mutants are used as templates for
subsequent rounds of mutagenesis/screening. In a knowledge-
based approach, a region within the active site of phenyl-
acetone monooxygenase (PAMO) from Thermobifida fusca
(ZP_57328)10 was modified to alter the substrate specificity
of the enzyme.11 This was possible on the basis of the
3-dimensional structure of PAMO constituting the first
crystal structure of a BVMO.12
against a library of structurally diverse ketones of different
polarity and electronic properties. In this contribution, we
present the most interesting results of a comparative study
of biotransformations by mutant enzymes and wild-type
CHMO (Scheme 1 and Table 1).
Recombinant whole-cell expression systems were utilized
to implement easy-to-use cofactor recycling (NADPH) and
biocatalyst production within living E. coli cells.9 We used
a screening methodology outlined recently based on a 24-
well plate format with a fermentation volume of 1 mL (0.5
mg of substrate).13 This multidish format reflects fermentation
characteristics of shake-flask cultures to a significant extent,
hence allowing also an assessment of biocatalyst efficiency
and performance on larger scale. In combination with chiral-
phase GC analysis, this screening method allows the
investigation of diverse ketones with respect to conversion
and stereoselectivity within a relatively short period of time.
Conversion of 5,5′-bicycloketone 1a with native CHMO
gave (-)-lactone 1b in very good yield and good enantio-
selectivity. Exchange of phenylalanine by serine at position
432 resulted in a slight increase in stereoselectivity compared
to wild-type CHMO, whereas mutations L426P and A541V
showed a significant decrease of enantiopreference. Remark-
ably, the more lipophilic chloro-bicyclic compound 2a was
biooxidized to enantiocomplementary lactone 2b with good
stereoselectivity, depending on the particular mutant used.
Again, mutation F432S resulted in the same stereoselectivity
as in wild-type CHMO, and mutations at positions 426 and
541 in this case led to formation of the antipodal lactone.
In the cyclobutanone series (substrates 3a, 4a) a related
trend was observed, although the degree of enantioselectivity
turned out to be lower. The mutation F432S for the more
polar precursor 3a led to the antipodal lactone 3b compared
to wild-type CHMO. A different mutation at position 432
(Phe f Ile) gave a slight increase in the enantioselectivity
compared to the wild-type enzyme. Similar results were
obtained with phenylcyclobutanone 4a. The mutation F432S
(together with K78E) increased the stereoselectivity signifi-
cantly compared to wild-type CHMO, while the mutation
F432I had the opposite effect resulting in almost racemic
lactone 4b.
While these previous studies aimed at the modification of
a specific property of the enzyme with respect to the
transformation of a particular substrate, in this contribution
the impact of mutations on a variety of substrates is
investigated. We selected a number of CHMO mutants that
displayed enantiocomplementary biooxidations of 4-hy-
droxycyclohexanone.9 These mutant enzymes were screened
(2) Mihovilovic, M. D.; Rudroff, F.; Gro¨tzl, B.; Kapitan, P.; Snajdrova,
R.; Rydz, J.; Mach, R. Angew. Chem., Int. Ed. 2005, 44, 3609.
(3) (a) Mihovilovic, M. D.; Rudroff, F.; Mu¨ller, B.; Stanetty, P. Bioorg.
Med. Chem. Lett. 2003, 13, 1479. (b) Wang, S.; Kayser, M. M.; Iwaki, H.;
Lau, P. C. K. J. Mol. Catal. B: Enzym. 2003, 22, 211. (c) Mihovilovic, M.
D.; Mu¨ller, B.; Kayser, M. M.; Stanetty, P. Synlett 2002, 700.
(4) (a) Mihovilovic, M. D.; Kapitan, P. Tetrahedron Lett. 2004, 45, 2751.
(b) Kelly, D. R.; Knowles, C. J.; Mahdi, J. G.; Taylor, I. N.; Wright, M. A.
J. Chem. Soc., Chem. Commun. 1995, 729. (c) Alphand, V.; Furstoss, R. J.
Org. Chem. 1992, 57, 1306.
(5) (a) Svendsen, A. Enzyme Functionality - Design, Engineering, and
Screening; Marcel Dekker: New York, 2005. (b) Brakmann, S.; Schwien-
horst, A. EVolutionary Methods in Biotechnology (CleVer Tricks for Directed
EVolution); Wiley-VCH: Weinheim, 2004. (c) Arnold, F. H.; Georgiou,
G. Directed EVolution: Screening and Selection Methods; Humana Press:
Totowa, NJ, 2003; Vol. 230.
Based on previous results for the enantiocomplementary
biooxidation of 4-hydroxycyclohexanone,9 we investigated
different substituted prochiral cyclohexanones 5a-7a. Sur-
prisingly, no enantiodivergence was observed, and neither a
change of hybridization (6a) nor modifications in polarity
(7a) of substituents at C-4 showed substantial influence on
the stereopreference. Stereoselectivity was slightly improved
for lactones 6b and 7b.17 The given results are representative
for a larger set of ketones investigated.
(6) Reetz, M. T. Angew. Chem., Int. Ed. 2001, 40, 284.
(7) (a) Reetz, M. T. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 5716. (b)
Reetz, M. T In AdVances in Catalysis; Gates, B. C., Kno¨tzinger, H., Eds.;
Elsevier: Amsterdam, 2005; Vol. 49, p 1.
(8) Donoghue, N. A.; Norris, D. B.; Trudgill, P. W. Eur. J. Biochem.
1976, 63, 175.
(9) Reetz, M. T.; Brunner, B.; Schneider, T.; Schulz, F.; Clouthier, C.
M.; Kayser, M. M. Angew. Chem., Int. Ed. 2004, 43, 4075.
(10) Fraaije, M. W., Kamerbeek, N. M.; Heidekamp, A. J.; Fortin, R.;
Janssen, D. B. J. Biol. Chem. 2004, 279, 3354.
(11) Bocola, M.; Schulz, F.; Leca, F.; Vogel, A.; Fraaije, M. W.; Reetz,
M. T. AdV. Synth. Catal. 2005, 347, 979.
(12) Malito, E.; Alfieri, A.; Fraaije, M. W.; Mattevi, A. Proc. Natl. Acad.
Sci. U.S.A. 2004, 101, 13157.
(13) Mihovilovic, M. D.; Snajdrova, R.; Winninger, A.; Rudroff F. Synlett
2005, 18, 2751.
(14) Mihovilovic, M. D.; Mu¨ller, B.; Kayser, M. M.; Stewart, J. D.;
Stanetty, P. Synlett 2002, 703.
(15) Gagnon, R.; Grogan, G.; Groussain, E.; Pedragosa-Moreau, S.;
Richardson, P. F.; Roberts, S. M.; Willetts, A. J.; Alphand, V.; Lebreton,
J.; Furstoss, R. J. Chem. Soc., Perkin Trans. 1 1995, 2527.
(16) Alphand, V.; Mazzini, C.; Lebreton, J.; Furstoss, R. J. Mol. Catal.
B: Enzym. 1998, 5, 219.
(17) Taschner, M.; Black, D. J. J. Am. Chem. Soc. 1988, 110, 6892.
1222
Org. Lett., Vol. 8, No. 6, 2006

Table 1. Screening Results of CHMO Mutants Produced by Directed Evolution9
a Sign of specific rotation. b Conversion: +++: >90%, ++: 50-90%, +: <50%. c Not applicable. d No conversion. e No baseline separation. f “Normal”
lactone. g “Abnormal” lactone. h Ratio between “normal” and “abnormal” lactones 12b.
In a number of previous studies it became apparent that
wild-type CHMO does not accept structurally demanding
ketones. Therefore, we focused our study on this class of
substrates and obtained very interesting results with exo
tricyclic ketones (8a, 9a). Due to multiple mutations, an
expansion in substrate acceptance was observed. The most
interesting mutations are L426P and A541V in mutant 1-H7-
F4 and various amino acid exchanges at position 432. The
saturated exo compound 8a gave 92% enantioselectivity with
mutant 1-C2-B7 and essentially racemic lactone with mutant
1-H7-F4 (12% ee). Interesting results were obtained with
the unsaturated exo-tricyclic ketone 9a, whereas the mutant
1-K2-F5, with a mutation at position 432, produced (-)-
lactone 9b with high enantioselectivity, and biooxidation with
mutant 1-H7-F4 resulted in formation of the antipodal (+)-
product.
In the endo-tricyclic series (10a, 11a), again mutations at
position 432 showed the strongest effects. The exchange of
phenylalanine by isoleucine resulted in a significant decrease
in enantioselectivity, whereas mutation F432S had no effect
on the stereopreference.
Finally, the regiodivergent biooxidation of a racemic fused
bicycloketone was investigated (12a) (Scheme 2). In general,
compound 12a was converted to both regioisomers (“normal”
Org. Lett., Vol. 8, No. 6, 2006
1223

substrate specificity and stereoselectivity. In particular,
mutations at position 432 have a significant impact on the
characteristics of mutant biocatalysts. The identification of
such hot spots offers the option of modifications of the
enzyme in subsequent rounds of biocatalyst optimization
either by rational design or directed evolution.7,18
Scheme 2. Regiodivergent Baeyer-Villiger Oxidation of
Ketone 12a: Formation of “Normal” and “Abnormal” Lactone
12b
However, as opposed to naturally evolved BVMOs,2 no
explicit trend was identified for the effect of specific amino
acid exchanges on both substrate acceptance and stereopref-
erence. This study accumulated a multitude of novel data
on structurally diverse substrates, which may be compiled
into a more comprehensive model on molecular level. So
far, this remains a major challenge, as previous studies
suggested major conformational changes of such enzymes
during the complete catalytic cycle.12 A homology model
of CHMO11 made possible by the X-ray structure of PAMO12
forms the basis of a future study aimed at understanding the
source of enhanced or inverted stereoselectivity.
and “abnormal” lactone; ratio 70/30) by wild-type CHMO
in very high optical purity for the abnormal lactone and
moderate stereoselectivity for the normal lactone. The two
mutants 1-H7-F4 and 1-K2-F5 displayed an increase in the
enantioselectivity of the “normal” lactone and by improving
the regioselectivity of the biooxidation toward the optimal
50:50 distribution.
In summary, we have demonstrated the potential of
random mutagenesis for the wide-spread modification of a
Baeyer-Villiger monooxygenase. This tool offers a powerful
methodology to optimize several properties of biocatalysts,
ultimately overcoming limitations which hampered the
widespread application of enzymes in synthetic chemistry
in the past.
Within this study, we could demonstrate that a limited
number of modifications within a protein can significantly
alter both substrate acceptance and stereopreference com-
pared to the wild-type enzyme. In a number of cases, new
substrates were identified and enantiocomplementary biooxi-
dations were realized.
Acknowledgment. Financial support by the Austrian
Science Fund (FWF, Project No. P 16373), by Vienna
University of Technology, and by the Fonds der Chemischen
Industrie (Frankfurt) is acknowledged.
Supporting Information Available: Details of the screen-
ing method and corresponding chiral GC data. This material
is available free of charge via the Internet at http://pubs.acs.org.
OL0601040
(18) CASTing has recently emerged as an alternative to traditional forms
of random mutagenesis: (a) Reetz, M. T.; Bocola, M.; Carballeira, J. D.;
Zha, D.; Vogel, A. Angew. Chem., Int. Ed. 2005, 44, 4192. (b) Reetz, M.
T.; Wang, L.-W. In part Bocola, M. Angew. Chem., Int. Ed. 2006, 15, 1236.
The results obtained also confirm the existence of certain
“hot spots” within CHMO of paramount influence to both
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Org. Lett., Vol. 8, No. 6, 2006