Structure-Based Site-Directed Mutagenesis of Hydroxynitrile Lyase from Cyanogenic Millipede, Oxidus gracilis for Hydrocyanation and Henry Reactions

Siriporn Chaikaew; Yukio Watanabe; Daijun Zheng; Fumihiro Motojima; Takuya Yamaguchi; Yasuhisa Asano

doi:10.1002/cbic.202400118

Structure-Based Site-Directed Mutagenesis of Hydroxynitrile Lyase from Cyanogenic Millipede, Oxidus gracilis for Hydrocyanation and Henry Reactions

Chembiochem. 2024 Mar 25:e202400118. doi: 10.1002/cbic.202400118. Online ahead of print.

Authors

Siriporn Chaikaew^{1

2}, Yukio Watanabe¹, Daijun Zheng^{1

2}, Fumihiro Motojima^{1

2}, Takuya Yamaguchi^{1

2}, Yasuhisa Asano¹

Affiliations

¹ Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama, 939-0398, Japan.
² Asano Active Enzyme Molecule Project, ERATO, JST.

PMID: 38526556
DOI: 10.1002/cbic.202400118

Abstract

Hydroxynitrile lyase (HNL) from the cyanogenic millipede Oxidus gracillis (OgraHNL) is a crucial enzyme in the cyanogenesis pathway. Here, the crystal structures of OgraHNL complexed with sulfate, benzaldehyde (BA), (R)-mandelonitrile ((R)-Man), (R)-2-chloromandelonitrile ((R)-2-Cl-Man), and acetone cyanohydrin (ACN) were solved at 1.6, 1.7, 2.3, 2.1, and 2.0 Å resolutions, respectively. The structure of OgraHNL revealed that it belonged to the lipocalin superfamily. Based on this structure, positive variants were designed to further improve the catalytic activity and enantioselectivity of the enzyme for asymmetric hydrocyanation and Henry reactions.

Keywords: Hydroxynitrile lyase; Oxidus gracilis; crystal structure; docking simulation; millipede; site–directed mutagenesis.

Abstract

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