Investigation of Structural Determinants for the Substrate Specificity in the Zinc‐Dependent Alcohol Dehydrogenase CPCR2 from Candida parapsilosis

Abstract

Zinc-dependent alcohol dehydrogenases (ADHs) are a class of enzymes applied in different biocatalytic processes ranging from lab to industrial scale. However, one drawback is the limited substrate range, necessitating a whole array of different ADHs for the relevant substrate classes. In this study, we investigated structural determinants of the substrate spectrum in the zinc-dependent ADH carbonyl reductase 2 from Candida parapsilosis (CPCR2), combining methods of mutational analysis with in silico substrate docking. Assigned active site residues were genetically randomized, and the resulting mutant libraries were screened with a selection of challenging carbonyl substrates. Three variants (C57A, W116K, and L119M) with improved activities toward different substrates were detected at neighboring positions in the active site. Thus, all possible combinations of the mutations were generated and characterized for their substrate specificity, yielding several improved variants. The most interesting were a C57A variant, with a 27-fold increase in specific activity for 4'-acetamidoacetophenone, and the double mutant CPCR2 B16-(C57A, L119M), with a 45-fold improvement in the kcat ⋅KM (-1) value. The obtained variants were further investigated by in silico docking experiments. The results indicate that the mentioned residues are structural determinants of the substrate specificity of CPCR2, being major players in the definition of the active site. Comparison of these results with closely related enzymes suggests that these might even be transferred to other ADHs.

Inversion of cpADH5 Enantiopreference and Altered Chain Length Specificity for Methyl 3‐Hydroxyalkanoates

“…Candida parapsilosis alcohol dehydrogenase 5 (cpADH5), formerly also named as Candida parapsilosis carbonyl reductase 2 (CPCR2), belongs to the zinc‐dependent medium chain alcohol dehydrogenases (MDRs) family . cpADH5 has a broad substrate range toward aliphatic ketones, keto esters, cyclic ketones, and aryl or aromatic substituted secondary alcohols . Yamamoto and co‐workers reported that cpADH5 is capable of oxidizing secondary alcohols such as ( S )‐2‐butanol, ( S )‐1‐phenylethanol, and achiral 2‐propanol .…”

“…Furthermore, cpADH5 has been investigated in detail by employing computational approaches. Carbonyl reduction mechanism as well as structural determinants of substrate spectrum was determined by using quantum mechanichs/molecular mechanics (QM/MM) techniques . Natural substrates and thus, function of the cpADH5 were predicted through virtual screening and QM/MM calculations .…”

“…Carbonyl reduction mechanism as well as structurald eterminants of substrate spectrum was determined by using quantum mechanichs/molecularm echanics (QM/MM) techniques. [17,21] Natural substrates and thus, function of the cpADH5 were predicted through virtuals creening and QM/MM calculations. [16a] In addition, its substrate binding pocket was engineered throughs tructure guided design forr eduction of aryl ketones such as 2-bromo-4'-chloroacetophenone.…”

Clarifying the Difference between Iterative Saturation Mutagenesis as a Rational Guide in Directed Evolution and OmniChange as a Gene Mutagenesis Technique

“…In the comparative study, CAST/ISM was tested in what we consider to be a questionable procedure, specifically when pitching it against the subsequent OmniChange experiments: Four single one‐residue sites were first chosen, three from a previous study (C57, W116 and L119), and one from a computational analysis (W286). Then conventional NNK‐based SM by using QuikChange was applied individually to all four single‐residue sites, as schematically illustrated in Figure B with B, C, E, and H representing the single residues.…”

A Comparative Reengineering Study of cpADH5 through Iterative and Simultaneous Multisite Saturation Mutagenesis

“…In this study we report a first comprehensive comparison of iterative and simultaneous site saturation mutagenesis for the example of improved cpADH5 activity. Three amino acid positions (Figure ; C57, W116 and L119) were selected from previous literature and one additional residue was selected from computational analysis (Figure ; W286). In total, four residues were subjected to simultaneous multiple site saturation or iterative recombination (exploring 17 paths) in order to improve the oxidation activity of cpADH5 toward methyl 3‐hydroxyhexanoate.…”