Expression, Structure Determination And Rational Design Of The Stereospecific Reductases From Candida Parapsilos

Oxidoreductase, a kind of biocatalyst with high stereoselectivity, is always used in bioconversion of optically active chiral compounds. Among various of stereospecific oxidoreductases from microorganisms, (R)- and (S)-specific carbonyl reductases from Candida parapsilosis CCTCC M203011 catalyze 2-hydroxyacetophenone to stereospecific 1-phenyl-1,2-ethanediol (PED). (R)-carbonyl reductase (RCR) catalyzes the reversible reaction between 2-hydroxyacetophenone and (R)-PED, and (S)-carbonyl reductase (SCR) catalyzes the reduction of 2-hydroxyacetophenone to (S)-PED. In order to further facilitate the two biocatalysts for stereoselective conversion and modify their biocatalytic functions, the coding sequences of RCR were optimized to improve its protein expression using genetic engineering methods. The crystal structure of SCR was also determined. Based on its refined three dimensional structure and catalytic functional domain, a series of mutants were rationally designed to change the cofactor specificity and interface characteristics using protein engineering technique. This work will supply the new research viewpoint for further understanding and modifying the catalytic functions of the enzyme.According to the preferred codons in Escherichia coli, nine rare codons were synonymously replaced with those used at higher frequency and the disorder sequence of 4-27 bp at 5'-terminus was truncaed in the RCR coding gene. The results showed that the total enzyme activity of the codon variant was increased 35.7% than before optimization and its protein production was also considerably improved. Atomic force microscopy images showed that the apo-enzyme adopted a netlike aggregate morphology at a high concentration. When the protein was complexed with NAD+, NADH or the ligand of 2-hydroxyacetophenone, most molecules of the protein resembled flat elliptical cylinders, which is similar to that of the enzyme at the low concentrations. By kinetic analysis the ratios of KM and kcat between RCR catalyzing 2-hydroxyacetophenone and (R)-PED were 0.5 and 1.8, respectively, and their ratio value of kcat/KM was about 4.0. The results showed that RCR catalyzed the NADH-linked reduction of 2-hydroxyacetophenone more easily than the NAD+-linked oxidation of (R)-PED.The RCR or SCR genes fused with enhanced green fluorescence protein (EGFP) were cloned and constructed on the eukaryotic expression vector pYX212. Using EGFP as specific biomarkers, two carbonyl reductases were mostly found to locate on the cell membrane, i.e. golgi complex endomembrane system, few dotted in the cells. According to the intensity of EGFP the level of SCR expression was found higher than RCR in Saccharomyces cerevisiae W303-1A cells. The fusion enzyme RCR-EGFP catalyzed the reduction of 2-hydroxyacetophenone to (R)-PED with the optical purity of 86.6% and yield of 70.4%, while the enzyme SCR-EGFP transformed the (S)-isomer with the optical purity of 92.3% in a yield of 81.8%. The results showed that the fusion of RCR or SCR with EGFP showed no effect in correct protein folding and physical catalytic functions.The 6×Histidine tag was inserted N terminus of RCR or SCR, and their fused genes were cloned using molecular recombinant technique and located downstream of the AOX1 promoter. The recombinant Pichia pastoris were successfully constructed. The highly heterogenous expressions of two carbonyl reductases were obtained. By analysis of the pre-expression and optimized expression process, the optimal culturing conditions were achieved as follows: pH 7.0, the initial OD600 2.0–2.5, methanol daily addition concentration of 1.0% (v/v) and the optimal induction time points at about 3.5–4.0 d for the strain GS115. Under these conditions, the productions of recombinant RCR and SCR were 185 mg/L and 270 mg/L in P. pastoris, the specific activity of both enzymes were 1.35 U/mg and 3.41 U/mg. The western blotting result with only one band appeared on polyvinylidene fluoride (PVDF) membrane revealed that Ab1 bound specifically to the 6×Histidine tag of RCR and SCR. The purity of both proteins was over 90% after a single Ni2+ -cherating purification step. The experiments showed that the optimal pH values for biotransfomation were 8–9 and 7.0 for RCR and SCR respectively. The purified RCR catalyzed the product (R)-PED with the optical purity of 83.7% and yield of 67.2%, while the enzyme SCR transformed (S)-isomer with the optical purity of 96.9% in a yield of 88.7%. The results also revealed that the addition of the"functional elements"Zn2+ of 5 mM considerably enhanced the biotransformation efficiency of (R)-PED, and the optical purity and yield of the product reached 94.3% and 86.5%.The RCR and SCR genes from C. parapsilosis were constructed an expression vector pETDuetTM-1 contain double cloning sites simultaneously and the recombinant plasmid pETDuet-RCR-SCR was obtained. Pyrimidine nucleotide transhydrogenase A and B genes from E. coli were inserted into the expression vector pACYCDuet-1 and the recombinant plasmid pACYD-PNTA-PNTB was constructed. The plasmids pETDuet-RCR-SCR and/or pACYD-PNTA-PNTB were then transformed the competent cells of E. coli BL21 (DE3), and the recombinant strains containing two or four target genes. The biotransformation experiments showed that strains catalyzed (R)-PED to (S)-isomer in one-step efficiently. The product was afforded with high optical purity of 96.3%e.e. and yield of 91.9%. When compared to the co-expression system containing RCR and SCR, the optical purity and yield was increased with 32% and 39.2%.The recombinant SCR was purified and crystallized, the crystal form was obtained using the hanging-drop vapor-diffusion method and reservoir solution of 18% (w/v) polyethylene glycol 2000 monomethyl ether and 8% (v/v) isopropyl as the precipitant. The crystals obtained after 5 d of growth were good enough for data collection and were rhomboid in shape with average dimensions of 0.3×0.3×0.4 mm. The crystal structure of mannitol 2-dehydrogenase (MtDH, PDB file 1H5Q) from Agaricus bisporus was used as a search model for molecular replacement. The crystal belongs to the space groups P212121 with cell dimensions of a= 104.7 ?; b= 142.8 ?; and c= 151.8 ?. The crystal structure of the apo-form was solved to a 2.69-? resolution to final Rwork of 20.5% (Rfree of 26.4%), with good stereochemical properties. Eight SCR molecules were identified per asymmetric unit and were organized into two tetramers, or eight SCR molecules in an asu form four dimmers, each is related by the so-called Q-axis dyad symmetry. Three novel features were found between the crystal structure of SCR and the other short-chain dehydrogenase/reductase members: (1) an extended N-terminal peptide tail which stabilizes the Q-axis related dimerization; (2) an unusual tetramerization which is devoid of 2-2-2 point-group symmetry, particularly in the R-axis related dimer interface; and (3) two of the four potential NADPH binding sites in the tetramer are occupied by surrounding peptides in the apo-enzyme form. Gel filtration experiments further suggests that a conformational change in the SCR subunits induced by NADPH binding converts the tetramer into an active form.A number of mutagenesis-enzymatic analyses were carried out based on the new structural information. The mutations of N-terminal peptide, the putative catalytic Ser-Tyr-Lys triad and key amino acids between interfaces were carried out. The results showed that the functional role of N-terminal peptide stabilizes the Q-axis related dimerization and has no any contribution to enzyme activity. SCR uses the same conserved catalytic triad as other SDR proteins. To investigate a possible functional role of the A-B interface, a dimer-breaking mutation in the dyad symmetrical hydrophobic interface was introduced. In particular, a tightly packed Val residue at position 270 in theβG strand was replaced with a negatively charged residue, Asp, which was expected to disrupt the A-B type dimerization without affecting the A-C interface. Consistent with our prediction, analytic ultracentrifugation measurements confirmed that the V270D mutant of SCR exists as a homo-dimer in solution. Kinetic studies indicate that SCR may maintain its enzymatic activity in a dimer form. Thermal stability of all the variants were analyzed using a circular dichroism temperature scan, resulting in a 45–48℃melting temperature, 4–7℃lower than that of the wild type SCR.For industrial applications, converting the cofactor specificity of an enzyme from NADPH to NADH would be of great significance as NADH is substantially cheaper than NADPH. Based on the determined quaternary structure of SCR, the mutations were rationally designed in order to explore the possibility of converting SCR from a NADPH-dependent enzyme into an NADH-dependent one. Using site-directed mutagenesis, the mutants were designed with different combinations of Ser67Asp, His68Asp and Pro69Asp substitutions inside or adjacent to the coenzyme binding pocket of so-called phosphate-binding loop betweenβB andαC. All mutations caused a significant shift of enantioselectivity toward the (R)-configuration during 2-hydroxyacetophenone reduction with different optical purity and yield. The mutant S67D/H68D produced (R)-PED with high opitical purity of 95.4% and yield of 83.1% in the NADH-linked reaction. By kinetic analysis, the mutant S67D/H68D resulted in a nearly 10-fold increase and a 20-fold decrease in the kcat/KM value when NADH and NADPH were used as the cofactors respectively, but maintaining kcat essentially the same with respect to wild-type SCR. The ratio of Kd values between NADH and NADPH for the S67D/H68D mutant and SCR were 0.28 and 1.9 respectively, which indicates the S67D/H68D mutant has a stronger preference for NADH and weaker binding for NADPH. Moreover, the S67D/H68D enzyme exhibited the similar secondary structure and melting temperature to the wild-type from circular dichroism analysis. It was also found that NADH provided maximal protection against thermal and urea denaturation for S67D/H68D, in contrast to the effective protection by NADP(H) for the wild-type enzyme. Thus, the double point mutation S67D/H68D converted the coenzyme specificity of SCR from NADP(H) to NAD(H) as well as the product enantioselectivity successfully without disturbing enzyme stability. The work provides a protein engineering approach to modify the co-enzyme specificity and enantioselectivity of ketone reduction for short-chain reductases and will likely have valuable industrial applications.