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.
Highlights
Stereoselective hydrolytic opening of epoxide rings is attractive in asymmetric synthesis, because the resultant vicinal diols are valuable building blocks of chiral drugs and bioactive compounds [1, 2]
Representative examples include the epoxide hydrolases (EHs) Nasvi-EH1 involved in the biosynthesis of an insect sex attractant [22], the a/b-EHs NcsF2 and SgcF-catalyzed epoxide ring opening in the biosynthesis of enediyne antitumor antibiotics neocarzinostatins and C-1027 [23, 24], the TsrI-mediated endopeptidyl hydrolysis and epoxide ring opening/macrocyclization in thiostrepton biosynthesis [25], and the Alp1U-catalyzed epoxide hydrolysis of epoxykinamycins (Fig. 1C) [26]
An atypical oxirane oxygen hole is identified in Alp1U, consisting of three residues (Trp-186/Trp-187/ Tyr-247) that are distinct from the Tyr/Tyr pair in classic a/b-EHs
Summary
Stereoselective hydrolytic opening of epoxide rings is attractive in asymmetric synthesis, because the resultant vicinal diols are valuable building blocks of chiral drugs and bioactive compounds [1, 2]. Single-crystal X-ray structures of the two regioselective Alp1U variants in complex with 1 were determined.
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