Mutation of an Atypical Oxirane Oxyanion Hole Improves Regioselectivity of the α/β-fold Epoxide Hydrolase Alp1U

Overview

Journal J Biol Chem

Specialty Biochemistry

Date 2020 Oct 2

PMID 33004437

Citations 3

Authors

Liping Zhang

Bidhan Chandra De

Wenjun Zhang

Attila Mandi

Zhuangjie Fang

Chunfang Yang

Yiguang Zhu

Tibor Kurtan

Changsheng Zhang

Affiliations

Soon will be listed here.

Abstract

Epoxide hydrolases (EHs) have been characterized and engineered as biocatalysts that convert epoxides to valuable chiral vicinal diol precursors of drugs and bioactive compounds. Nonetheless, the regioselectivity control of the epoxide ring opening by EHs remains challenging. Alp1U is an α/β-fold EH that exhibits poor regioselectivity in the epoxide hydrolysis of fluostatin C (compound 1) and produces a pair of stereoisomers. Herein, we established the absolute configuration of the two stereoisomeric products and determined the crystal structure of Alp1U. A Trp-186/Trp-187/Tyr-247 oxirane oxygen hole was identified in Alp1U that replaced the canonical Tyr/Tyr pair in α/β-EHs. Mutation of residues in the atypical oxirane oxygen hole of Alp1U improved the regioselectivity for epoxide hydrolysis on 1. The single site Y247F mutation led to highly regioselective (98%) attack at C-3 of 1, whereas the double mutation W187F/Y247F resulted in regioselective (94%) nucleophilic attack at C-2. Furthermore, single-crystal X-ray structures of the two regioselective Alp1U variants in complex with 1 were determined. These findings allowed insights into the reaction details of Alp1U and provided a new approach for engineering regioselective epoxide hydrolases.

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