title: 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.
authors.affiliation: Institute for Microbiology, Chair for Molecular Biotechnology, Technische Universität Dresden, Zellescher Weg 20b, 01217 Dresden (Germany).
authors.forename: Christoph
authors.initials: C
authors.lastname: Loderer
authors.affiliation: Department of Biotechnology, RWTH Aachen University, Worringerweg 1, 52074 Aachen (Germany).
authors.forename: Gaurao V
authors.initials: GV
authors.lastname: Dhoke
authors.affiliation: Department of Biotechnology, RWTH Aachen University, Worringerweg 1, 52074 Aachen (Germany).
authors.forename: Mehdi D
authors.initials: MD
authors.lastname: Davari
authors.affiliation: Institute of Chemistry, University of Graz, NAWI Graz, Heinrichstrasse 28, 8010 Graz (Austria).
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Kroutil
authors.affiliation: Department of Biotechnology, RWTH Aachen University, Worringerweg 1, 52074 Aachen (Germany).
authors.forename: Ulrich
authors.initials: U
authors.lastname: Schwaneberg
authors.affiliation: Department of Biotechnology, RWTH Aachen University, Worringerweg 1, 52074 Aachen (Germany).
authors.forename: Marco
authors.initials: M
authors.lastname: Bocola
authors.affiliation: Institute for Microbiology, Chair for Molecular Biotechnology, Technische Universität Dresden, Zellescher Weg 20b, 01217 Dresden (Germany). marion.ansorge@tu-dresden.de.
authors.forename: Marion B
authors.initials: MB
authors.lastname: Ansorge-Schumacher
mesh_terms: D000098:Acetophenones / Q000737:chemistry / Q000378:metabolism; D000426:Alcohol Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002175:Candida / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D004252:DNA Mutational Analysis; D062105:Molecular Docking Simulation; D017354:Point Mutation; D011487:Protein Conformation; D013379:Substrate Specificity; D015032:Zinc / Q000378:metabolism*
chemical_list: D000098:Acetophenones; D000426:Alcohol Dehydrogenase; D015032:Zinc
keywords: biocatalysis; enzyme catalysis; molecular docking; oxidoreductases; protein engineering; structure-activity relationships