6KXH

Alp1U_Y247F mutant in complex with Fluostatin C

Summary for 6KXH

• Entry DOI: 10.2210/pdb6kxh/pdb
• Related: 6kxc
• Descriptor: Putative hydrolase, Fluostatin C, D-MALATE, ... (5 entities in total)
• Functional Keywords: hydrolyse epoxide, cis-vicinal diol, hydrolase
• Biological source: Streptomyces ambofaciens (strain ATCC 23877 / 3486 / DSM 40053 / JCM 4204 / NBRC 12836 / NRRL B-2516)
• Total number of polymer chains: 4
• Total formula weight: 149773.94

Authors

Zhang, L.,Yingli, Z.,De, B.C.,Zhang, C. (deposition date: 2019-09-11, release date: 2020-09-16, Last modification date: 2024-03-27)

Primary citation

Zhang, L.,De, B.C.,Zhang, W.,Mandi, A.,Fang, Z.,Yang, C.,Zhu, Y.,Kurtan, T.,Zhang, C.
Mutation of an atypical oxirane oxyanion hole improves regioselectivity of the alpha / beta-fold epoxide hydrolase Alp1U.
J.Biol.Chem., 295:16987-16997, 2020

PubMed 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.

PubMed: 33004437
DOI: 10.1074/jbc.RA120.015563

Experimental method

X-RAY DIFFRACTION (1.78039550258 Å)