Epoxide ring opening reactions are common and important in both biological processes and synthetic applications and can be catalyzed in a non-redox manner by epoxide hydrolases or reductively by oxidoreductases. Here we report that fluostatins (FSTs), a family of atypical angucyclines with a benzofluorene core, can undergo nonenzyme-catalyzed epoxide ring opening reactions in the presence of flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NADH). The 2,3-epoxide ring in FST C is shown to open reductively via a putative enol intermediate, or oxidatively via a peroxylated intermediate with molecular oxygen as the oxidant. These reactions lead to multiple products with different redox states that possess a single hydroxyl group at C-2, a 2,3-vicinal diol, a contracted five-membered A-ring, or an expanded seven-membered A-ring. Similar reactions also take place in both natural products and other organic compounds harboring an epoxide adjacent to a carbonyl group that is conjugated to an aromatic moiety. Our findings extend the repertoire of known flavin chemistry that may provide new and useful tools for organic synthesis.
Title: Mutation of an atypical oxirane oxyanion hole improves regioselectivity of the alpha/beta-fold epoxide hydrolase Alp1U Zhang L, De BC, Zhang W, Mandi A, Fang Z, Yang C, Zhu Y, Kurtan T, Zhang C Ref: Journal of Biological Chemistry, 295:16987, 2020 : PubMed
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 alpha/beta-fold EH that exhibits poor regioselectivity in the epoxide hydrolysis of fluostatin C (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 W186/W187/Y247 oxirane oxygen hole was identified in Alp1U that replaced the canonical Tyr/Tyr pair in alpha/beta-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, while 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.
