Improvement of enantioselectivity of the B-type halohydrin hydrogen-halide-lyase from Corynebacterium sp. N-1074

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• 作者：Fumiaki Watanabe¹ ; Fujio Yu¹ ; Akashi Ohtaki² ; Yasuaki Yamanaka² ; Keiichi Noguchi³ ; Masafumi Odaka² ; ⁴ ; Masafumi Yohda² ; ^{yohda@cc.tuat.ac.jp" class="auth_mail" title="E-mail the corresponding author}
• 关键词：Enantioselectivity ; Halohydrin ; Crystal structure ; Lyase
• 刊名：Journal of Bioscience and Bioengineering
• 出版年：2016
• 出版时间：September 2016
• 年：2016
• 卷：122
• 期：3
• 页码：270-275
• 全文大小：1070 K

文摘

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.