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Applied and Environmental Microbiology, June 2007, p. 3759-3764, Vol. 73, No. 11
0099-2240/07/$08.00+0     doi:10.1128/AEM.02185-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

Purification, Characterization, Gene Cloning, and Expression of a Novel Alcohol Dehydrogenase with Anti-Prelog Stereospecificity from Candida parapsilosis ^,

Yao Nie, Yan Xu,^* Xiao Qing Mu, Hai Yan Wang, Ming Yang, and Rong Xiao

Key Laboratory of Industrial Biotechnology of Ministry of Education and School of Biotechnology, Southern Yangtze University, Wuxi 214122, People's Republic of China

Received 16 September 2006/ Accepted 31 March 2007

Top ABSTRACT INTRODUCTION Nucleotide sequence accession... REFERENCES

 
An alcohol dehydrogenase from Candida parapsilosis CCTCC M203011 was characterized along with its biochemical activity and structural gene. The amino acid sequence shows similarity to those of the short-chain dehydrogenase/reductases but no overall identity to known proteins. This enzyme with unusual stereospecificity catalyzes an anti-Prelog reduction of 2-hydroxyacetophenone to (S)-1-phenyl-1,2-ethanediol.

Top ABSTRACT INTRODUCTION Nucleotide sequence accession... REFERENCES

 
Alcohol dehydrogenases (EC 1.1.1.X [where X ranges from 1 to 28]) belong to a class of oxidoreductases and catalyze the NAD(P)H-dependent reduction of a variety of endogenous and xenobiotic carbonyl compounds (32). There is considerable interest in the use of alcohol dehydrogenases for the production of chiral alcohols in the pharmaceutical and fine chemicals industries (28, 33, 34). For the inherent advantages over chemocatalysts in terms of their high chemo-, enantio-, and regioselectivity, stereospecific alcohol dehydrogenases are very interesting from both scientific and industrial perspectives (18).

Alcohol dehydrogenases for asymmetric reduction are ubiquitous in nature and have been characterized from diverse sources, including bacteria (10), yeasts (17), plants (23), and tissues from several mammalian species (37). They display different physical and enzymatic properties, show a wide variety of substrate specificities, and are mainly classified into three superfamilies, zinc-dependent alcohol dehydrogenases, short-chain dehydrogenase/reductases (SDRs), and aldo-keto reductases, based on their catalytic properties and sequence information (8, 14, 32). Among the various enzyme sources, Candida species are attractive as highly stereospecific oxidoreductase donors (4, 13, 15, 16, 25, 30). In many cases, the stereospecific oxidoreductases from strains of the genus Candida are dissimilar in structure and classified in different superfamilies (15, 16, 38).

To date, however, most enzymes catalyzing reductions generally follow Prelog's rule in the sense of the stereochemistry outcomes (2, 31), and other types of biocatalysts with a complementary selectivity are yet limited. Only a few microorganisms were found to possess anti-Prelog selectivity, such as Geotrichum spp. (36), Yarrowia lipolytica (5), Lactobacillus brevis (6), Lactobacillus kefiri (3), and Pseudomonas spp. (2). Moreover, few enzymes with unusual, anti-Prelog stereoselectivity have been isolated and characterized in purified forms, and the corresponding amino acid sequences and biophysical parameters remain unknown. To our knowledge, only the R-specific alcohol dehydrogenase from Lactobacillus brevis (LB-RADH) was analyzed for its primary structure and further crystal structure (26). Because of the scarcity of enzymes with anti-Prelog selectivity, however, the precise mechanism of enzymatic stereopreference in asymmetric reduction is not yet fully understood. Therefore, the discovery of enzymes catalyzing the anti-Prelog-type reaction would be valuable not only for filling the demands for asymmetric synthesis but also for providing a research basis for clarifying the relationship between protein structure and stereospecificity, which is useful in the alteration of stereospecificity by the approach of site-directed evolution (9).

In a previous study, we found that Candida parapsilosis CCTCC M203011 efficiently catalyzed the deracemization of racemic 1-phenyl-1,2-ethanediol (PED) to an S-enantiomer (24), which is a versatile chiral building block in organic synthesis (11, 21). This reaction process of stereoinversion involves two steps, the oxidation step of (R)-PED to the intermediate (2-hydroxyacetophenone) by an R-specific alcohol dehydrogenase and the reduction step of the intermediate to (S)-PED (24). The second step of asymmetric reduction is an anti-Prelog-type reaction involving an alcohol dehydrogenase with unusual stereospecificity (CPADH) (2, 22) (see Fig. S1 in the supplemental material). In this report, we describe the purification, characterization, gene cloning, and expression of the alcohol dehydrogenase from C. parapsilosis and also analyze the primary structure of CPADH.

The microorganism C. parapsilosis CCTCC M203011 from the China Center for Type Culture Collection (CCTCC, Wuhan, China) was incubated as described previously (24). Then, the culture (100 ml), grown for 24 hours, was used as an inoculum in a 5-liter fermentor containing 3 liters of medium. After cultivation for 48 h, the cells (about 150 g) were harvested for enzyme purification. The alcohol dehydrogenase of C. parapsilosis was purified by column chromatographic procedures, including the use of DEAE-Sepharose, phenyl-Sepharose, and Blue Sepharose, following the ammonium sulfate precipitation (see the supplemental material). Enzyme activity was measured as described previously (24), and protein concentration was determined using the Bradford method (1). The enzyme was purified about 660-fold over the activity of the cell extract, with an activity yield of 2.9%, and the specific activity of the final enzyme preparation was 99 U/mg (see Table S1 in the supplemental material). The relative molecular mass of the native enzyme was determined to be 30 kDa by gel filtration on a Protein KW-803 (Shodex, Japan) column, and the purified enzyme showed a single band with a molecular mass of 31 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with 12% polyacrylamide gels (19) (see Fig. S2 in the supplemental material), indicating that this enzyme has a monomeric structure and is smaller than the carbonyl reductase, a dimer of 135 kDa, from C. parapsilosis (29).

The optimal pH of the enzyme was determined with a pH range of 3.5 to 9.0 and using the following buffers: 0.1 M acetate (pH 3.5 to 6.0), 0.1 M potassium phosphate (pH 6.0 to 8.0), and 0.1 M Tris-HCl (pH 8.0 to 9.0). The pH stability of the enzyme was performed by measuring the residual activity for 2-hydroxyacetophenone reduction after conservation in the pH series described above. The enzyme was the most active at pH 4.5 and stable in a pH range of 4.0 to 8.0 when kept at 4°C for 48 h (Fig. 1A). The optimum temperature for the enzyme was 35°C, among temperatures ranging from 20 to 70°C, and the enzyme was stable at up to 40°C when heated at various temperatures for 60 min (Fig. 1B). The effects of several metal ions (CaCl₂, CoCl₂, CuSO₄, FeSO₄, MgSO₄, MnSO₄, NiCl₂, and ZnSO₄) and inhibitor EDTA on the enzyme activity were investigated in a final concentration of 1 mM by using 2-hydroxyacetophenone as the substrate in the reaction. The enzyme tolerated a range of divalent transition metal ions, except for Cu²⁺, which inhibited the enzyme activity, with a loss of 99%. The metal-chelating reagent EDTA did not influence the enzyme activity, suggesting that the enzyme is metal ion independent and dissimilar to the stereoselective oxidoreductases containing an essential metal ion of Zn²⁺ or Mg²⁺ (3, 39).

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FIG. 1. Effects of pH and temperature on the activity and stability of CPADH. (A) Effects of pH on enzyme activity (•) and stability (). (B) Effects of temperature on enzyme activity () and stability (). The enzyme activities for 2-hydroxyacetophenone reduction were measured as described in the text. Maximal enzyme activity observed was set as 100% relative activity.

The substrate specificity of the purified CPADH was investigated with various carbonyl compounds and (R,S)-PED (Table 1). The enzyme had high specificity to 2-hydroxyacetophenone and ethyl 4-chloroacetoacetate with NADPH as the coenzyme. However, the activities of the enzyme for other alkyl and aromatic ketones were relatively low. These results suggested that CPADH is NADPH dependent and specific towards the carbonyl compounds with a substitute group, such as hydroxyl and halogen, neighboring the carbonyl group. The reversibility of the enzymatic reaction was also investigated with PED as the substrate and NADP⁺ or NAD⁺ as the coenzyme. There is no oxidative activity detected, suggesting that the thermodynamic behaviors of PED and 2-hydroxyacetophenone are not favorable for the oxidation of PED and that the equilibrium of the reaction between PED and 2-hydroxyacetophenone is towards PED, which results from the Gibbs-free energy difference between the alcohol and the carbonyl compound. Aside from being tested in an enzyme activity assay, CPADH was further evaluated for catalyzing asymmetric reduction in a reaction mixture (2 ml) comprising 0.1 M potassium phosphate buffer (pH 6.5), 0.5 g/liter 2-hydroxyacetophenone, and NADPH (10 µmol). The reaction was carried out at 30°C for 8 h with shaking. The resulting products were analyzed as described previously (24). Optically pure PED, S-enantiomer, was detected (>99% enantiomeric excess) when 2-hydroxyacetophenone was reduced by CPADH (Fig. 2). The stereochemical outcome of CPADH catalyzing 2-hydroxyacetophenone reduction to give (S)-PED indicated an anti-Prelog-type reaction (22). Therefore, CPADH with unusual, anti-Prelog-stereospecificity would necessarily supplement the stereospecific oxidoreductases described to date in the catalysis of the reduction of prochiral carbonyl compounds to the corresponding optically pure alcohols in an anti-Prelog reaction type. As with CPADH, in our previous research, an R-specific alcohol dehydrogenase, which catalyzes the first step of oxidizing (R)-PED to 2-hydroxyacetophenone in deracemization, was isolated from C. parapsilosis (24). The discrepancies in both purification steps and enzyme properties indicate that different oxidoreductases acting on the same carbonyl compound may exist in one microorganism cell.

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TABLE 1. Substrate specificity of CPADHa

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FIG. 2. Asymmetric reduction of 2-hydroxyacetophenone by using purified CPADH. mAU, milli-absorbance units. (A) Standard samples (R)-PED (retention time, 15.0 min) and (S)-PED (retention time, 18.3 min). (B) 2-Hydroxyacetophenone without enzyme (retention time, 27.1 min). (C) Reaction products.

The N-terminal amino acid sequencing of CPADH was first attempted by the automated Edman degradation method but was not successful, suggesting that the N terminus of CPADH might be blocked. Therefore, trypsin digestion of CPADH and analysis of the resulting peptides by liquid chromatography-tandem mass spectrometry (LC-MS-MS) was attempted. Tryptic peptides were obtained by in-gel digestion in 50 mM ammonium bicarbonate overnight at 37°C after reduction and alkylation as described by Sleat et al. (35). Proteolytic digests of purified CPADH reconstituted in 0.1% formic acid were analyzed by nanospray LC-MS-MS using an LTQ linear ion trap (ThermoElectron) and a Micromass Q-TOF API US (Waters) mass spectrometer (see the supplemental material). The sequences of two fragments were identified as NVLDLFSLK (CPADH-1) and WWQLTPLGR (CPADH-2). Based on these amino acid sequences, degenerate primers were designed as follows: forward primer, 5'-AAYGTNYTNGAYYTNTT-3'; and reverse primer, 5'-ARNGGNGTNARYTGCCACC-3' (with Y representing C or T; N representing A, C, G, or T; H representing A, C, or T; and R representing A or G). To determine the complete nucleotide sequence, genomic DNA was digested with EcoRI, circularized by ligation, and subjected to inverse PCR with the gene-specific primers 5'-TGGGCACCATTTGCTAGAGTGAAC-3' and 5'-CGAATGGACCCCATATGTCTTTTG-3'. The complete nucleotide sequence of the CPADH gene (cpadh) contains one complete open reading frame with a length of 840 bp, encoding a polypeptide of 279 amino acid residues. The molecular mass of the deduced polypeptide was calculated to be 30,088 Da. The deduced amino acid sequence of CPADH was aligned using a BLAST search tool (http://www.ncbi.nlm.nih.gov/BLAST/). The similarity search results revealed a high identity of CPADH with a hypothetical protein from Candida albicans SC5314 (76%), a carbonyl reductase S1 from Candida magnoliae (55%), an NADP-dependent mannitol dehydrogenase from Cladosporium fulvum (50%), an L-xylulose reductase from Hypocrea jecorina (49%), an NADP-dependent mannitol dehydrogenase from Davidiella tassiana (49%), a mannitol dehydrogenase from Botryotinia fuckeliana (48%), a short-chain dehydrogenase/reductase from Aspergillus fumigatus Af293 (42%), a carbonyl reductase from Kluyveromyces aestuarii (40%), a short-chain dehydrogenase/reductase from Chromohalobacter salexigens DSM 3043 (40%), a short-chain dehydrogenase/reductase from Thermotoga maritima MSB8 (39%), a short-chain dehydrogenase/reductase from Kineococcus radiotolerans SRS30216 (38%), and a short-chain dehydrogenase/reductase from Deinococcus geothermalis DSM 11300 (37%) (Fig. 3). The conserved sequences of the SDR superfamily, i.e., the cofactor-binding motif Gly-X-X-X-Gly-X-Gly (X denotes any amino acid) and a common Ser-X_n-Tyr-X-X-X-Lys sequence in active sites, were found as Gly41, Gly45, Gly47, Ser174, Tyr187, and Lys191 in CPADH (27). These highly conserved sequences indicate that CPADH belongs to the family of classical SDRs and extends to the cP2 subfamily, which is one of the three NADPH-dependent subfamilies (12). The catalytic triad of Ser-Tyr-Lys has also been extended to a tetrad of Asn-Ser-Tyr-Lys in the majority of SDRs, and the highly conserved and catalytically critical asparagine residue was also observed in CPADH as Asn146 (7). In the alignment of similar proteins (Fig. 3), there are some strictly conserved residues in the C-terminal parts of CPADH and some mannitol dehydrogenases, which would reflect the substrate specificity of SDRs (27). Thus, CPADH may act on mannitol and play a role in mannitol metabolism in C. parapsilosis (20). Among other stereospecific oxidoreductases from C. parapsilosis mainly classified into the zinc-dependent alcohol dehydrogenases and the aldo-keto reductases (16, 29), CPADH is the first stereospecific SDR discovered from C. parapsilosis strains. Furthermore, the amino acid sequence of CPADH does not show strict identity to those of any proteins of known function, and thus the enzyme was proposed to be a novel alcohol dehydrogenase with anti-Prelog stereospecificity.

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FIG. 3. Amino acid sequence alignment of CPADH from C. parapsilosis (CpADH), hypothetical protein from C. albicans SC5314 (CaHP) (GenBank accession no. EAK96529), carbonyl reductase S1 from C. magnoliae (CmCR) (GenBank accession no. BAB21578), an NADP-dependent mannitol dehydrogenase from Cladosporium fulvum (CfMDH) (GenBank accession no. AAK67169), an L-xylulose reductase from Hypocrea jecorina (HjXR) (GenBank accession no. AAM20896), an NADP-dependent mannitol dehydrogenase from Davidiella tassiana (DtMDH) (GenBank accession no. AAO91801), a mannitol dehydrogenase from Botryotinia fuckeliana (BfMDH) (GenBank accession no. ABC84216), a short-chain dehydrogenase/reductase from Aspergillus fumigatus Af293 (AfSDR) (GenBank accession no. EAL91661), a carbonyl reductase from Kluyveromyces aestuarii (KaCR) (GenBank accession no. BAD01116), a short-chain dehydrogenase/reductase from Chromohalobacter salexigens DSM 3043 (CsSDR) (GenBank accession no. ABE59960), a short-chain dehydrogenase/reductase from Thermotoga maritima MSB8 (TmSDR) (GenBank accession no. AAD35385), a short-chain dehydrogenase/reductase from Kineococcus radiotolerans SRS30216 (KrSDR) (GenBank accession no. EAM74531), and a short-chain dehydrogenase/reductase from Deinococcus geothermalis DSM 11300 (DgSDR) (GenBank accession no. ABF44294). Gaps in the aligned sequences are indicated by dashes. Identical amino acid residues are enclosed in boxes. Amino acids matching those identified by partial amino acid sequence analysis by LC-MS-MS are underlined.

A CPADH expression plasmid (pETCPADH) was constructed by inserting cpadh into pET-21c (Novagen). The sense primer 5'-ATCGGATCCGATGGGCGAAATCGAATCTTATTG-3', containing a BamHI restriction site (underlined), and the antisense primer 5'-TGACTCTCGAGTGGACACGTGTATCCACCGTC-3', containing an XhoI restriction site (underlined), were synthesized. The purified PCR-amplified products digested with BamHI and XhoI were ligated into the BamHI-XhoI restriction sites of pET-21c, and Escherichia coli BL21(DE3) was transformed with the ligation mixture to yield the transformant E. coli BL21(DE3)(pETCPADH). The heterologous expression was induced with 0.1 mM IPTG (isopropyl-ß-D-thiogalactopyranoside) when the culture turbidity at 600 nm was 0.6. After cultivation for 12 h at 30°C, the activities of the recombinant enzyme were measured using cell extracts. The 2-hydroxyacetophenone-reducing activities of the E. coli BL21(DE3) cells transformed with pETCPADH were 2.58 U/mg under IPTG induction and 0.13 U/mg without IPTG induction. Furthermore, a predominant band corresponding to the expected size of the recombinant enzyme (31 kDa) was also observed in the soluble fraction of induced cells (see Fig. S3A in the supplemental material). The overexpressed enzyme constituted about 30% of the total soluble proteins in the intracellular fraction. The recombinant C-terminal His₆-tagged protein was purified from the cell extract of E. coli transformants by using a HisTrap HP affinity column, following the protocol provided by the manufacturer (GE Healthcare), to an apparent homogeneity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (see Fig. S3B in the supplemental material). This recombinant CPADH displayed physical characteristics similar to those observed for the native enzyme from C. parapsilosis. By use of the purified recombinant enzyme, optically pure PED of S-configuration (>99% enantiomeric excess) was obtained from the asymmetric reduction of 2-hydroxyacetophenone.

The discovery of the alcohol dehydrogenase catalyzing anti-Prelog-type reactions and the profound knowledge of the molecular biology of the novel enzyme could have advantages not only for meeting various demands of asymmetric synthesis and understanding the diversity of various oxidoreductases with dissimilar characteristics in microorganisms but also for providing a research basis for elucidating the relationship between protein three-dimensional structures involving key amino acid residues in the active site and its stereopreference for catalyzing asymmetric reactions, which is valuable for the creation of an enzyme with a desired stereospecificity by the approach of site-directed evolution (9). Apart from the scientific interest in the molecular mechanism of alcohol dehydrogenase catalyzing asymmetric reactions, the overexpressed CPADH would be a potential catalyst for the industrial application of producing chiral alcohols.

Top ABSTRACT INTRODUCTION Nucleotide sequence accession... REFERENCES

 
The nucleotide sequence of the cpadh gene from C. parapsilosis CCTCC M203011 has been deposited in the GenBank database under accession number DQ675534.

 
The financial support of the National Natural Science Foundation of China (grant no. 20376031), the National Key Basic Research and Development Program of China (973 Program) (grant no. 2003CB716008), Ministry of Education, People's Republic of China, under the Program for New Century Excellent Talents in University (grant NCET-04-0498), and the Program for Changjiang Scholars and Innovative Research Team in University (grant IRT0532) is gratefully acknowledged.

 
* Corresponding author. Mailing address: Key Laboratory of Industrial Biotechnology of Ministry of Education and School of Biotechnology, Southern Yangtze University, 1800 Lihu Rd., Wuxi 214122, People's Republic of China. Phone: 86-510-85864735. Fax: 86-510-85864112. E-mail: yxu{at}sytu.edu.cn

Published ahead of print on 13 April 2007.

Supplemental material for this article may be found at http://aem.asm.org/.

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Applied and Environmental Microbiology, June 2007, p. 3759-3764, Vol. 73, No. 11
0099-2240/07/$08.00+0     doi:10.1128/AEM.02185-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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