Acta Biochim Biophys Sin 2009, 41: 163–170
Cloning, expression and characterization of a novel diketoreductase from Acinetobacte baylyi
Laboratory of Chemical Biology, School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China
*Corresponding author: Tel, 86-25-85391150; Fax, 86-25-83271249; E-mail, firstname.lastname@example.org
Reductions of carbonyl groups catalyzed by oxidoreductases are involved in all biological processes, and are often a class of important biocatalyst. In this paper, we reported a novel enzyme designated as diketoreductase that was able to reduce two carbonyl groups in a diketo ester to corresponding dihydroxy ester with excellent stereoselectivity. The diketoreductase was cloned from Acinetobacter baylyi by reverse genetic method, heterogeneously expressed in Escherichia coli and purified to homogeneity by two chromatographic steps. This novel enzyme exhibited dual cofactor specificity, with a preference of NADH over NADPH. The dihydroxy ester product catalyzed by the diketoreductase was only 3R,5S-stereoisomer with both diastereomeric excess and enantiomeric excess values more than 99.5%. In addition, some biochemical properties of the enzyme, such as the optimal pH and temperature, were also characterized. Furthermore, sequence analysis indicated that this new enzyme was homologous to bacterial 3-hydroxyacyl coenzyme-A dehydrogenase. More importantly, based on the unique catalytic activity and excellent stereoselectivity, the diketoreductase could be utilized in the synthesis of valuable chiral drug intermediates, such as Lipitor®.
Keywords diketoreductase; oxidoreductase; stereoselectivity; chiral alcohols; statin
Received: October 18, 2008 Accepted: November 16, 2008
Ketoreductases, an abundant group of oxidoreductase, are present in various bacteria, yeast and fungi. They commonly participate in many biological processes in all living organisms . Due largely to the capability of producing chiral alcohols and high enantioselectivity, ketoreductases have been recognized and utilized as an important class of versatile biocatalysts in the chemical and pharmaceutical industries for the preparation of chiral intermediates [2-9].
Introduction of a chiral center in a substrate with single or multiple carbonyl groups can be easily accomplished by different ketoreductases with NADH or NADPH as a cofactor . For example, Lactobacillus brevis converts the δ- keto group of a β, δ-diketo ester to its δ-hydroxy product [10,11]. However, the stereoselective reduction of two carbonyl groups in the same molecule to form a chiral β, δ-dihydroxy product by a single enzyme has not been evidently characterized. Previously, screening of microorganisms with ethyl-6-(benzyloxy)-3,5-dioxohexanoate (1) revealed that an Acinetobacter species was able to reduce both β and δ-carbonyl groups of the substrate to its 3R,5S-ethyl-6-(benzyloxy)-3,5-dihydroxyhexanoate (2) product with 93.2% enantiomeric excess (ee) and 63.3% diastereomeric excess (de), and an enzyme possibly responsible for the conversion was isolated and preliminarily studied [12,13]. However, the identity and detailed characterization of such an interesting enzyme has not been further investigated.
In present study, the gene encoding a diketoreductase (DKR), catalyzing double reduction of a β,δ-diketo ester as shown in Fig. 1, was cloned from Acinetobacter baylyi ATCC 33305 and expressed in Escherichia coli in a soluble form. After purification of the recombinant enzyme, the properties of the enzyme, in terms of the catalytic activity and stereoselectivity, were thoroughly characterized. It is expected that the unique reaction catalyzed by DKR with unprecedented stereoselectivity could be useful for the synthesis of chiral intermediates, particularly the chiral dihydroxy hexanoate chains of synthetic statin drugs.
Materials and Methods
Strains and chemicals
A. baylyi ATCC 33305
was obtained from American Type Culture Collection (ATCC). pET
22b (+) expression vector was purchased from Novagen
Microorganism and culture
The culture of A. baylyi ATCC
33305 is maintained in our laboratory as frozen
vials at -
Cloning of DKR
The genomic DNA was
extracted from A. baylyi
ATCC 33305 by a standard phenol-chloroform method. Degenerate PCR primers based
on the partial amino acid sequences of the N-terminus TGITNVTV (Oligo No. 1, 5'-ACIGGIATIACIAAYGTIACIGTI-3') and an
internal peptide GELAPAK (Oligo No. 2,
5'-GGIGARCTRGCICCIGCIAAR-3') of the previously purified ketoreductase
from Acinetobacter species , where “R” and “Y”
are A+G and C+T, were used to amplify the gene from A. baylyi ATCC 33305 genomic DNA. The
amplification condition was as follows:
Heterologous expression of DKR
To facilitate the ligation of the DKR gene with expression vector pET22b (+) (Novagen), oligonucleotide primers were prepared to include 5'- and 3'-terminals of the gene along with compatible restriction cleavage sites:
Oligo 3 (5'-GATACGTGCCATATGACCGGCATCACGAATGTCACCGTTCTC-3'; 5'-terminal of the
site underlined) and Oligo 4 (5'-GATACGTGCAGATACGTGCAGGATCCTCAGTACCGGTAGAAGCCCTCGCC-3'; 3'-terminal of the
site underlined). PCR reaction was performed with Oligo
3 and Oligo 4 as primers and the 5.2 kb KpnI fragment as
template under the same thermo cycling conditions as described above. Following
agarose gel electrophoresis, an approximately 900 bp PCR fragment was amplified and purified using Axyprep DNA gel extraction kit (
The pET22b (+)-DKR construct was transformed into E.
coli BL21 (DE3) by CaCl2. Transformed cells were selected on LB-ampicillin agar medium, and the resulting transformants were cultured in 50 ml LB medium containing 100 mg/ml ampicillin at
Purification of recombinant DKR
Frozen E. coli cells (
Spectrophotometric method was used to determine the DKR activity on a
UV-1700 array spectrophotometer () whose cell compartment was maintained at
Determination of native molecular weight
The Sephadex G-100 column was employed to determine the molecular weight of DKR. The calibration curve of proteins with standard molecular weight was obtained by using gel filtration calibration kits from Sigma according to the manufacture’s protocol.
Optimal pH and temperature
The optimal pH and temperature for
the reduction of 1 catalyzed by DKR
were determined by spectrophotometric method. For pH
optima, assays were conducted at
To determine the
kinetic constants for the reaction of 1 with
NADH, reaction mixtures contained
Bioconversion of 1 and product identification
50 ml reaction was performed to examine the bioconversion of 1 to 2. The reaction mixture contained 73 mg (0.26 mmoles) of
Cloning and expression of DKR
Screening of different microorganisms with 1 revealed that A. baylyi ATCC 33305 was able to convert 1 to 2 with high stereoselectivity. Therefore, a PCR product of approximately 350 bp was obtained using the primers based on the N-terminal and internal peptide sequences reported in the literature  and the genomic DNA extracted from A. baylyi ATCC 33305. The cDNA fragment labeled with digoxigenin-dUTP was used as a probe to hybridize the genomic DNA library prepared with different endonucleases digestions. After sequencing, an 852 bp gene with an open reading frame encoding 283 amino acid residues (Fig. 2; GenBank accession Nos. EU273886, ABY48099) was obtained from a 5.2 kb fragment of KpnI digestion. Blast search showed that DKR is highly homologous to 3-hydroxyacyl coenzyme-A dehydrogenase (HACD) from various sources [15-17], and the homolog with a putative HACD gene from Nocardia farcinica IFM 10152 (GenBank accession No. YP 120052) is as high as 78% .
the expression of DKR, restriction sites of
BamHI and NdeI were
introduced into the DKR gene in order
to express a recombinant protein with the original sequence. The DKR gene
was ligated into pET22b (+) expression vector, and
subsequently transformed to BL21 (DE3) E.
coli strain for expression. By comparing
different induction conditions, expression level of the recombinant protein
reached maximum at 12 h analyzed by SDS-PAGE after IPTG addition [Fig. 3(A)], and majority of the protein was successfully expressed in
the soluble form with 50 mM IPTG at
Purification and properties of DKR
After expression, the cells containing DKR were harvested, disrupted and centrifuged. The resulting supernatant was purified to homogeneity judged by SDS-PAGE [Fig. 3(B)] by DEAE-Sepharose and Sephadex G-100 chromatography steps. Furthermore, the molecular weight of DKR was determined as 30 kDa by Sephadex G-100 gel filtration, indicating the native protein exists as a monomer based on the calculated molecular mass of 30,278.4 Da.
The basic properties of the purified DKR were evaluated.
DKR showed a relatively broad pH optimum centered at pH 6.0 using
Because a common catalytic feature of ketoreductases is the requirement of a hydride donor [2-5], usually NADH or NADPH, the preliminary steady state kinetic experiments with DKR were performed to compare and assess the cofactor preference by spectrophotometric assays at the optimal conditions. The kinetic constants were obtained by analyzing the experimental data with reciprocal Lineweaver-Burk method. As shown in Table 1, DKR has dual cofactor specificity, which is similar to other ketoreductases , but NADH was much more favorable and efficient than NADPH as a cofactor for DKR because the catalytic efficiency of NADH was three orders of magnitude higher than NADPH.
Catalytic activity of DKR and its stereoselectivity
To identify the reaction catalyzed by recombinant DKR, bioconversion was performed using 1 as a substrate in the presence of the NADH regeneration system . After extraction and purification, the product was characterized by 1H NMR and mass spectrometry. As a result, the spectral data of the product were in agreement with 2 reported in the literature . Moreover, the stereoselectivity of DKR was evaluated by determining de and ee values of the isolated product with a chiral HPLC method. Fig. 4(A) shows a HPLC chromatogram of four stereoisomers produced by chemical reduction of 1. Compared to Fig. 4(A), the product from DKR catalysis only showed single 3R,5S-enantiomer with both de and ee values greater than 99.5% [Fig. 4(B)]. Such unprecedented diastereo- and enantioselectivity exhibited by DKR is extremely unusual.
Stereoselective reduction of one carbonyl group in a ketone molecule to from corresponding hydroxy product by ketoreductases is quite common in biological systems [2-11], but diketo-reduction in the same molecule with multiple carbonyl groups by single enzyme has not been evidently reported. When two carbonyl groups in the same molecule are reduced by chemical method, four stereoisomers are equally produced [Fig. 4(A)]. In the present study, when a β, δ-diketo ester substrate was reduced by DKR, only one 3R,5S-stereoisomer (2) appeared as shown in Fig. 4(B), which indicates that DKR may represent a new class of enzyme.
3-Hydroxyacyl coenzyme-A dehydrogenase (HACD) is a well studied enzyme that catalyzes the reduction of 3-oxoacyl-CoA to 3-hydroxyacyl-CoA [20-23]. The structure, catalytic mechanism and properties from various sources have been extensively reported [24-26], but the capability of reducing two carbonyl groups in the same molecule by HACD remains unknown. Although the Blast search showed that sequence of DKR is similar to a variety of HACD with high homology (between 30 and 78%) at nucleic acid and amino acid levels [15-17], all sequences in the databases with high homology are putative proteins without known function. Thus, it is unclear whether DKR is functionally related with HACD. Besides sequence alignments, clarification on the actual relationship of these enzymes with a series of experiments becomes interesting and important for the further investigation on this novel diketoreductase.
Generally, A. baylyi ATCC 33305 has been regarded as a pathogenic organism, but little is known on the mechanism of its virulence [27-30]. So far, secondary metabolism in this organism also remains unexplored. On the basis of present study, it is reasonable to speculate that DKR may play a role in the biosynthesis of secondary metabolites in this organism , which will be another subject of further study.
Statins, especially synthetic statins, such as Atorvastatin (3) and Rosuvastatin (4) (Fig. 1), are the top-selling cholesterol-lowering drugs in the world. However, because of the current complex and expensive route for synthesizing the chiral side chains, biocatalytic production of chiral side chain of statins has become a highly competitive area in which a number of approaches and routes have been reported [32-34]. Due to the exactly same stereochemistry of 2 compared to the chiral side chain of statins, the unique characteristics of DKR, in terms of the catalytic activity and the excellent stereoselectivity, make this enzyme as an attractive biocatalyst for the development of a practical, efficient and economic process for the synthesis of statin drugs.
In conclusion, a novel oxidoreductase designated as diketoreductase (DKR) has been successfully cloned, expressed and purified to homogeneity. Reduction of two carbonyl groups in the same molecule catalyzed by DKR and the stereoselectivity have also been investigated, indicating that DKR displays unique and rare characteristics. Because of the stereoselectivity and the reaction type, DKR could be utilized in the asymmetric synthesis of chiral drug intermediates, such as statin drugs. It is expected that exploration of DKR by detailed kinetics analyses and structure-function study may provide new insights on the novel catalytic mechanism.
We are grateful for the technical assistance from Jianhua Chen, Yan Huang, Xiaomei Liu, Dekang Liu and Jin Deng.
work is supported by the grants from the Start-up Fund from
1 Ikeda M, Kanao Y, Yamanaka M, Sakuraba H, Mizutani Y, Igarashi Y, Kihara A. Characterization of four mammalian 3-hydroxyacyl-CoA dehydratases involved in very long-chain fatty acid synthesis. FEBS Lett 2008, 582: 2435–2440.
2 Oppermann U. Carbonyl reductases: the complex relationships of mammalian carbonyl- and quinone-reducing enzymes and their role in physiology. Annu Rev Pharmacol Toxicol 2007, 47: 293-322.
3 Matsunaga T, Shintani S, Hara A. Multiplicity of mammalian reductases for xenobiotic carbonyl compounds. Drug Metab Pharmacokinet 2006, 21: 1-18.
4 Penning TM, Drury JE. Human aldo-keto reductases: function, gene regulation, and single nucleotide polymorphisms. Arch Biochem Biophys 2007, 464: 241-250.
5 Ellis EM. Reactive carbonyls and oxidative stress: potential for therapeutic intervention. Pharmacol Ther 2007, 115: 13-24.
6 Müller M, Wolberg M, Schubert T, Hummel W. Enzyme-catalyzed regio- and enantioselective ketone reduction. Adv Biochem Eng Biotechnol 2005, 92: 261-287.
7 Schmid A, Dordick JS, Hauer B, Kiener A, Wubbolts M, Witholt B. Industrial biocatalysis today and tomorrow. Nature 2001, 409: 258-268.
8 Schoemaker HE, Mink D, Wubbolts MG. Dispelling the myths- biocatalysis in industrial synthesis. Science 2004, 299: 1694-1697.
9 Kroutil W, Mang H, Edegger K, Faber K. Recent advances in biocatalytic reduction of ketones and oxidation of sec-alcohols. Curr Opin Chem Biol 2004, 8: 120-126.
10 Wolberg M, Hummel W, Wandrey C, Müller M. Highly regio- and enantioselective reduction of 3, 5-dioxocarboxylates. Angew Chem Int Ed 2000, 39: 4306-4308.
11 Wolberg M, Hummel W, Müller M. Biocatalytic reduction of beta, delta-diketo esters: a highly stereoselective approach to all four stereoisomers of a chlorinated beta, delta-dihydroxy hexanoate. Chem Eur J 2001, 21: 4562-4571.
12 Patel RN, Banerjee A, McNamee CG, Brzozowski D, Hanson RL, Szarka LJ. Enantioselective microbial reduction of 3, 5-dioxo-6-(benzyloxy) hexanoic acid, ethyl ester. Enzyme Microb Technol 1993, 15: 1014-1021.
13 Guo ZW, Chen YJ, Goswami A, Hanson RL, Patel R.N. Synthesis of ethyl and t-butyl (3R, 5S)-dihydroxy-6-benzyloxy hexanoates via diastereo- and enantioselective microbial reduction. Tetrahedron: Asymmetry 2006, 17: 1589-1602.
14 Liu JH, Yu BY, Chen YJ. Determination of enantiomeric excess of ethyl 3, 5- dihydroxy -6-benzyloxy hexanoate by chiral reverse phase high performance liquid chromatography. Chirality 2008, 25: 51-53.
15 Ishikawa J, Yamashita A, Mikami Y, HoshinoY, Kurita H, Hotta K, Shiba T et al. The complete genomic sequence of Nocardia farcinica IFM 10152. Proc Natl Acad Sci USA 2004, 101: 14925-14930.
16 Takarada H, Sekine M, Kosugi H, Matsuo Y, Fujisawa T, Omata S, Kishi E et al. Complete genome sequence of the soil actinomycete Kocuria rhizophila. J Bacteriol 2008, 190: 4139-4146.
17 White O, Eisen JA, Heidelberg JF, Hickey EK, Peterson JD, Dodson RJ, Haft DH et al. Genome sequence of the radioresistant bacterium Deinococcus radiodurans R1. Science 1999, 286: 1571-1577.
18 Kim DY, Stauffacher CV, Rodwell VW. Dual coenzyme specificity of Archaeoglobus fulgidus HMG-CoA reductase. Protein Sci 2000, 9: 1226-1234.
19 Geertman JM, van Dijken JP, Pronk JT. Engineering NADH metabolism in Saccharomyces cerevisiae: formate as an electron donor for glycerol production by anaerobic, glucose-limited chemostat cultures. FEMS Yeast Res 2006, 6: 1193-1203.
20 Schifferdecker J, Schulz H. The inhibition of L-3-hydroxyacyl-CoA dehydrogenase by acetoacetyl-CoA and the possible effect of this inhibitor on fatty acid oxidation. Life Sci 1974, 14: 1487-1492.
21 Holden HM, Banaszak LJ, Frieden C, McLoughlin DJ. Differences in the binding of coenzyme to L-3-hydroxyacyl-coenzyme A dehydrogenase in the crystalline state and in solution. FEBS Lett 1981, 132: 15-8.
22 Holden HM, Banaszak LJ. L-3-hydroxyacyl coenzyme A dehydrogenase. The location of NAD binding sites and the bilobal subunit structure. J Biol Chem 1983, 258: 2383-2389.
23 Hartmann D, Philipp R, Schmade1 K, Birktoft JJ, Banaszak LJ, Trammer WE. Spatial arrangement of coenzyme and substrates bound to L-3-hydroxyacyl-coA dehydrogenase as studied by spin-labeled analogues of NAD+ and CoA. Biochemistry 1991, 30: 2782-2790.
24 Haapalainen AM, van Aalten DMF, Meriläinen
G, Jalonen JE, Pirilä P, Wierenga RK, Hiltunen JK et al.
25 Powell AJ, Read JA, Banfield MJ, Gunn-Moore F, Yan SD,Lustbader J, Stern AR et al. Recognition of structurally diverse substrates by type II 3-hydroxyacyl-CoA dehydrogenase (HADH II)/amyloid-b binding alcohol dehydrogenase (ABAD). J Mol Biol 2000, 303: 311-327.
26 Birktoft JJ, Holden HM, Hamlin R, Xuong NH, Banaszak LJ. Structure of
L-3- hydroxyacyl- coenzyme A dehydrogenase:
preliminary chain tracing at 2.8-Å resolution. Proc Natl Acad Sci
28 Porstendörfer D, Gohl O, Mayer F, Averhoff B. A pilin-like protein essential for natural competencein Acinetobacter sp. strain BD413: regulation, modification, and cellular localization. J Bacteriol 2000, 182: 3673-3680.
29 Gohl O, Friedrich A, Hoppert M, Averhoff B. The thin pili of Acinetobacter sp. strain BD413 mediate adhesion to biotic and abiotic surfaces. Appl Environ Microbiol 2006, 72: 1394-1401.
30 Beceiro A, Perez-Llarena FJ, Perez A, Tomas MdM, Fernández A, Mallo S, Villanueva R, Bou G. Molecular characterization of the gene encoding a new AmpC beta-lactamase in Acinetobacter baylyi. J Antimicrob Chemother 2007, 59: 996-1000.
32 Thayer AM. Competitors want to get a piece of Lipitor. Chem Eng News 2006, 84: 26- 27.
33 Müller M. Chemoenzymatic synthesis of building blocks for statin side chain. Angew Chem Int Ed 2005, 44: 362-365.
34 Öhrlein R, Baisch G. Chemo-enzymatic approach to statin side-chain building blocks. Adv Synth Catal 2003, 345: 713-715.