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Mutation of an atypical oxirane oxyanion hole improves regioselectivity of the α/β-fold epoxide hydrolase Alp1U.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2020 Dec 11; Vol. 295 (50), pp. 16987-16997. Date of Electronic Publication: 2020 Oct 01. - Publication Year :
- 2020
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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.<br />Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.<br /> (© 2020 Zhang et al.)
- Subjects :
- Epoxide Hydrolases genetics
Hydrolysis
Kinetics
Mutagenesis, Site-Directed methods
Protein Structure, Tertiary
Sequence Homology, Amino Acid
Stereoisomerism
Structure-Activity Relationship
Crystallography, X-Ray methods
Epoxide Hydrolases chemistry
Epoxide Hydrolases metabolism
Epoxy Compounds chemistry
Ethylene Oxide chemistry
Mutation
Streptomyces enzymology
Subjects
Details
- Language :
- English
- ISSN :
- 1083-351X
- Volume :
- 295
- Issue :
- 50
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 33004437
- Full Text :
- https://doi.org/10.1074/jbc.RA120.015563