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Acta Biochim Biophys Sin 2009, 41: 163170

doi: 10.1093/abbs/gmn019.

Cloning, expression and characterization of a novel diketoreductase from Acinetobacte baylyi

 

Xuri Wu, Nan Liu, Yunmian He, and Yijun Chen*

 

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, yjchen@cpu.edu.cn

 

Abstract

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

 

Introduction

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 [1]. 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 [10]. 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-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 (Gibbstown, USA). NADH, NADPH, NAD+ and isopropyl β-D-1-thiogalactopyranoside (IPTG) were obtained from Sigma (St. Louis, USA). All chemicals and solvents were analytical grade. Ethyl 3,5-diketo-6-benzyloxyhexanoate (1), racemic ethyl 3,5-dihydroxy-6-benzyloxy hexanoate and ethyl 3R,5S-dihydroxy-6-benzyloxy hexanoate (2) were prepared according to the procedures described in the literature [13].

 

Microorganism and culture

The culture of A. baylyi ATCC 33305 is maintained in our laboratory as frozen vials at -70°C. Half milliliter of frozen A. baylyi ATCC 33305 culture was inoculated into 50 ml of LB medium consisting of 10 g peptone, 5 g yeast extract, 10 g NaCl per liter of water in a 250 ml flask and grown on a rotary shaker at 28°C, 220 rpm for 48 h. Ten milliliters of culture was used as an inoculum for the growth of cultures in one liter flask containing 200 ml of LB medium. The culture was grown at 28°C, 220 rpm for 24 h, and harvested by centrifugation at 13,500 g for 15 min.

 

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 [13], 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: 94°C for 4 min (1 cycle); 94°C for 1 min, 50°C for 1 min, 72°C for 2 min (30 cycles); 72°C for 7 min (1 cycle). The resulting approximately 350 bp PCR product was purified from agarose gel and ligated into cloning vector pTOPO-TA (Novagen) for sequencing. To obtain the gene with full length, the cDNA fragment was labeled with digoxigenin (Roche Applied Science, Basel, Switzerland). The labeled cDNA was then used as a probe to hybridize the genomic DNA library prepared with different endonuclease digestions.

 

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 gene, NdeI site underlined) and Oligo 4 (5'-GATACGTGCAGATACGTGCAGGATCCTCAGTACCGGTAGAAGCCCTCGCC-3'; 3'-terminal of the gene; BamHI 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 (Shenzhen, China). The PCR product (2 mg) was digested with the restriction enzymes and 2 mg of the expression vector pET22b (+) was similarly cleaved with these enzymes in parallel. The digested DNA fragments were electrophoresed on a 1.0% TAE agarose gel for 1.5 h at 90 V and purified using Axyprep DNA gel extraction kit. Then, the digested PCR fragment and pET22b (+) were ligated at a 5:1 molar ratio using a Fast link kit (Axyprep) at room temperature. The constructed pET22b (+) vector with desired insert was confirmed by electrophoresis and DNA sequencing.

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 37°C, 220 rpm for 20 h. The culture was diluted into fresh LB medium containing 100 mg/ml ampicillin with an OD600 of 0.30 and incubated under the same conditions with OD600=0.9. IPTG was added to final concentrations of 0.05 mM, 0.2 mM and 1 mM from a sterile 1 M stock in H2O at 15°C, 220 rpm to compare the expression levels. Samples of 1 ml each were taken at intervals of 2 h from 0 to 14 h after induction, and centrifuged at 13,500 g for 5 min. The cells were treated by SDS-loading buffer for SDS-PAGE analysis. For large scale expression, after 14 h of induction, the cells were pelleted by centrifugation (5000 g) for 15 min, resuspended in 0.5 volume of 0.1 M potassium phosphate buffer (pH 7.0) and centrifuged to collect cell pellets and store at –70°C for future uses.

 

Purification of recombinant DKR

Frozen E. coli cells (2.0 g) were suspended in 10 ml of 50 mM potassium phosphate buffer (pH 7.0) containing 1 mM DTT and 5% glycerol. The cell suspensions were passed through a high pressure cell press at 1000 psi for 5 min to disrupt the cells. Cell-free extract was obtained by centrifuging the mixture at 13,500 g for 30 min. The cell-free extract (10 ml) was applied onto a DEAE-Sepharose fast flow (GE Healthcare Biosciences, Wisconsin, USA) column (2.0 cm×20 cm) equilibrated with 50 mM potassium phosphate buffer (pH 8.0) containing 1 mM DTT and 5% glycerol. After equilibration, the column was eluted with 200 ml of a linear gradient of 0–0.5 M NaCl. Fractions with 3 ml each were collected at a flow rate of 1.0 ml/min. Active fractions (30 ml) were pooled, concentrated and desalted using an Amicon YM-10 membrane to final volume of 2 ml. Next, approximately 2 ml of the concentrated enzyme solution was loaded to the Sephadex G-100 (GE Healthcare Biosciences) column (1.0 cm×21 cm) and eluted with 50 mM potassium phosphate buffer (pH 7.0) containing1 mM DTT, 0.1 M NaCl and 5% glycerol at a flow rate of 0.5 ml/min. The active fractions were collected and assayed by spectrophotometric method. The purity of the enzyme was judged by SDS-PAGE, and protein concentration was determined by Bradford method.

 

Enzyme assay

Spectrophotometric method was used to determine the DKR activity on a UV-1700 array spectrophotometer (Shimadzu, Kyoto, Japan) whose cell compartment was maintained at 40°C during the measurements of absorbance change at 340 nm for the oxidation of NADH or NADPH. Assay mixture contained 0.15 mM NADH or NADPH, 0.25 mM 1, 550 mg DKR and 0.1 M potassium phosphate buffer (pH 6.0) in a final volume of 1.0 ml. The decrease of absorbance at 340 nm was monitored by the addition of enzyme. The reaction was linear in 2 min. For all assays, enzyme activity was defined as one unit representing the oxidation of one mmole of NADH or NADPH per minute per milligram protein.

 

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 30°C in a final volume of 1.0 ml using NADH as a cofactor. Sodium acetate buffer (0.1 M) was used for the pH range of pH 4.0 to 6.0, and 0.1 M potassium phosphate buffer for pH 6.0 to 8.0. For temperature optima, the reactions were conducted at pH 6.0 in a final volume of 1.0 ml at different temperatures ranging from 20°C to 50°C at intervals of 5°C. Specific activity of the enzyme was compared at different pH and temperatures to obtain the optimal reaction conditions.

 

Kinetic studies

To determine the kinetic constants for the reaction of 1 with NADH, reaction mixtures contained 0.15 mM NADH, various concentrations of 1, 0.05 U DKR and 0.1 M potassium phosphate buffer (pH 6.0) in a final volume of 1.0 ml. To determine the kinetic constants for the reaction of 1 with NADPH as a cofactor, assay mixtures contained 0.15 mM NADPH, various concentrations of 1, 0.05 U DKR and 0.1 M potassium phosphate buffer (pH 6.0) in a final volume of 1.0 ml. To determine the kinetic constants for NADH, reaction mixtures contained 0.25 mM 1, various concentrations of NADH, 0.05 U DKR and 0.1 M potassium phosphate buffer (pH 6.0) in a final volume of 1.0 ml. To determine the kinetic constants for NADPH, reaction mixtures contained 0.25 mM 1, various concentrations of NADPH, 0.05 U DKR and 0.1 M potassium phosphate buffer (pH 6.0) in a final volume of 1.0 ml.

 

Bioconversion of 1 and product identification

A 50 ml reaction was performed to examine the bioconversion of 1 to 2. The reaction mixture contained 73 mg (0.26 mmoles) of 1 in 2.5 ml ethanol, 0.5 mM NAD+, 200 units of formate dehydrogenase, 200 mM sodium formate, 47.5 units of enzyme solution and 0.1 M potassium phosphate buffer (pH 6.0). The reaction was initiated by addition of the DKR, and incubated at 28°C, 200 rpm for 18 h. Subsequently, the reaction was stopped and extracted with ethyl acetate (3 ml×25 ml). The organic phase was combined and evaporated to dryness by a rotary evaporator. The optical purity of yellow oil product was analyzed by chiral reverse phase high performance liquid chromatography as described by Liu et al [14]. The product identification was performed by 1H NMR spectroscopy and mass spectrometry.

 

Results

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 [13] 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% [15].

For 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 15°C, 220 rpm as shown in Fig. 3(B).

 

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 0.1 M potassium phosphate buffer by comparing the specific enzyme activity at different pH values and sodium acetate buffer (pH 6.0) gave compatible activity. The broad temperature optimum for DKR centered at 40°C was observed by comparing specific activity at pH 6.0 with different temperatures.

 

Cofactor requirement

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 [18], 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 [19]. 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 [13]. 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.

 

Discussion

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 [31], 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.

 

Acknowledgements

We are grateful for the technical assistance from Jianhua Chen, Yan Huang, Xiaomei Liu, Dekang Liu and Jin Deng.

 

Funding

This work is supported by the grants from the Start-up Fund from China Pharmaceutical University (No. 01211085), the “111 Project” from the Ministry of Education of China and the State Administration of Foreign Expert Affairs of China (No. 111-2-07).

 

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