Bioinformatic and biochemical analysis uncovers novel activity in the 2-ER subfamily of OYEs.

Abstract

Members of the old yellow enzyme (OYE) family utilize a flavin mononucleotide cofactor to catalyze the asymmetric reduction of activated alkenes. The 2-enoate reductase (2-ER) subfamily are of particular industrial relevance as they can reduce α/β alkenes near electron-withdrawing groups. While the broader OYE family is being extensively explored for biocatalytic applications, oxygen sensitivity and poor expression yields associated with the presence of an Fe/S cluster in 2-ERs have hampered their characterization. Herein, we explore the use of pseudo-anaerobic preparation as a route to more widespread study of these enzymes and apply bioinformatics approaches to identify a subset of 2-ERs containing unusual mutations in both a key catalytic residue and the Fe/S cluster-binding motif. Biochemical analysis of a representative member from Burkholderia insecticola (OYEBi) reveals novel N-methyl-proline demethylation activity, which we hypothesize may play a role in osmotic stress regulation based on genomic neighborhood analysis.