Molecular Engineering And Assessment Of Stereospecific Carbonyl Reductase From Candida Parapsilosis By Site-directed Mutagenesis

By homology modeling analysis, P124 and W125, the sites in cofactor-binding domain of the carbonyl reductase SCR1 from Candida parapsilosis were found to have a steric effect on the binding of NADPH. The site-directed mutagenesis of W125 and double mutation of P124 and W125 were performed to investigate the influence of these two sites on the catalytic activity of the enzyme. Additionally, Detection of protein-ligand interactions by NMR, combined with the analysis of the model structure of SCR1–NADPH, was performed to guide us in finding K138, which is in the substrate-binding domain of SCR1.(1) Based on the X-ray structure of alcohol dehydrogenase from Candida parapsilosis (CPADH, PDB code: 3ctm), we constructed SCR1 model structure and found W125 in cofactor-binding domain forming a loop which produced a steric effect on the binding of NADPH, as compared with other alcohol dehydrogenase. Then P124 and W125 were substituted for A and G, the amino acid residues with smaller side chain. W125A, and the resulted mutants W125G, P124A/W125A, P124G/W125G were constructed by site-directed mutation. Detection of protein-ligand interactions by NMR using reductive methylation of lysine residues, combined with the analysis of the model structure of SCR1–NADPH, was performed to identify K138 as the mutation target, which located in the substrate-binding domain of SCR1. Site-saturation mutagenesis of K138 was performed to investigate the influence of different amino acids residues in this site on the catalytic activity, substrate specificity and stereoselectivity of the enzyme.(2) We assayed the catalytic activity of the mutants toward model substrates 2-hydroxyacetophenone (2-HAP) and ethyl 4-chloro-3-oxobutyrate (COBE). The results showed that all mutants had low activity, illustrating the structure conformation of cofactor-binding domain changed. We also investigated the substrate specificity of the mutants towards aromatic ketone,β-ketone ester, and aliphatic ketones, respectively. The results showed that SCR1 showed activity toward 2-hydroxyacetophenone, 2-bromoacetophenone and its derivatives, and COBE, while the catalytic efficiency of mutants toward acetophenone and its derivatives and 2-octanone was enhanced. The mutant P124A/W125A showed highest activity toward valerophenone, suggesting that W125 affects the substrate recognition of enzyme. On the other hand, the stereospecificity of some mutants was even found to be inverted. The mutants P124A/W125A and P124G/W125G exhibited a shift of enantioselectivity toward 2-hydroxyacetophenone and ethyl 4-chloro-3-oxobutanoate to give the product in (R)-configuration.(3) A library of mutants including 19 mutants was created by means of site-saturation mutagenesis at residue K138 in the substrate-binding domain of SCR1, where the substituted residues were divided into basic amino acids, acidic amino acids, neutral amino acids, and nonpolar amino acids according to the nature of their side chains. We investigated the properties of the mutants library, involving the activity and the stereoselectivity toward model substrates 2-HAP and COBE, respectively, and the substrate specificity toward aromatic ketone,β-ketone ester, and aliphatic ketones. It can be seen that the relative activities of enzyme with basic amino acid residue at 138 were higher than others. Especially, the relative activities of K138H/R were higher than the wild-type enzyme SCR1.Then we measured the substrate specificity of the purified K138H/R toward substrate spectrum. The results showed that K138H exhibited higher specific activity than SCR1 toward most of substrates, especially for 2-HAP and COBE, and had a wider substrate spectrum. While K138R showed higher substrate specificity to the model substrates and maintained similar specific activity of SCR1 to other substrates. We also determined the stereoselectivity of mutants toward model substrates. of the library, most mutants favored the formation of (S)-configurated chiral alcohols for reduction of 2-HAP, while the ee value of K138R/H was higher than 99%. However, K138R possessed the highest enantioselectivity in the reduction of COBE, with 95% ee. K138E/I/T exhibited inverted enantioselectivity in the reduction of COBE, with 76% ee, 78% ee, and 75% ee, respectively. K138L maintained most activity of SCR1 and produced (S)-CHBE with ee value of 100%.