title: Improvement of enantioselectivity of the B-type halohydrin hydrogen-halide-lyase from Corynebacterium sp. N-1074.
abstract: Halohydrin hydrogen-halide-lyase (H-Lyase) is a bacterial enzyme involved in the degradation of halohydrins. This enzyme catalyzes the intramolecular nucleophilic displacement of a halogen by a vicinal hydroxyl group in halohydrins, producing the corresponding epoxides. The H-Lyases have been classified into A, B and C subtypes based on amino acid sequence similarities. These enzymes have attracted much attention as industrial catalysts in the synthesis of chiral chemicals from prochiral halohydrins. In the present study, we constructed mutants of B-type H-Lyase from Corynebacterium sp. N-1074 (HheB) displaying higher enantioselectivity by structure-based site-directed mutagenesis and random mutagenesis. A triple mutant of HheB exhibited 98.5% enantioselectivity, the highest ever reported, toward (R)-4-chloro-3-hydroxy-butyronitrile production, with the yield reaching approximately two-fold that of the wild-type enzyme. We discuss the structural basis of the high enantioselectivity and productivity of the mutant by comparing the crystal structures of the mutant HheB and the wild-type enzyme in complex with or without the substrate analogue.
authors.affiliation: Yokohama Research Laboratories, Mitsubishi Rayon Co. Ltd., Yokohama, Kanagawa 230-0053, Japan.
authors.forename: Fumiaki
authors.initials: F
authors.lastname: Watanabe
authors.affiliation: Yokohama Research Laboratories, Mitsubishi Rayon Co. Ltd., Yokohama, Kanagawa 230-0053, Japan.
authors.forename: Fujio
authors.initials: F
authors.lastname: Yu
authors.affiliation: Department of Biotechnology and Life Science, Graduate School of Technology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan.
authors.forename: Akashi
authors.initials: A
authors.lastname: Ohtaki
authors.affiliation: Department of Biotechnology and Life Science, Graduate School of Technology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan.
authors.forename: Yasuaki
authors.initials: Y
authors.lastname: Yamanaka
authors.affiliation: Instrumentation Analysis Center, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan.
authors.forename: Keiichi
authors.initials: K
authors.lastname: Noguchi
authors.affiliation: Department of Biotechnology and Life Science, Graduate School of Technology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan; Department of Life Science, Faculty and Graduate School of Engineering and Resource Science, Akita University, Akita City, Akita 010-8502, Japan.
authors.forename: Masafumi
authors.initials: M
authors.lastname: Odaka
authors.affiliation: Department of Biotechnology and Life Science, Graduate School of Technology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan. Electronic address: yohda@cc.tuat.ac.jp.
authors.forename: Masafumi
authors.initials: M
authors.lastname: Yohda
mesh_terms: D000595:Amino Acid Sequence; D055162:Biocatalysis; D003352:Corynebacterium / Q000201:enzymology* / Q000235:genetics; D018360:Crystallography, X-Ray; D004852:Epoxy Compounds / Q000378:metabolism; D008190:Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D004852:Epoxy Compounds; D050505:Mutant Proteins; D008190:Lyases; C066813:haloalcohol hydrogen-halide lyase
keywords: Crystal structure; Enantioselectivity; Halohydrin; Lyase