A redox-regulated, heterodimeric NADH:cinnamate reductase in Vibrio ruber

Abstract

Genes of putative reductases of ,{beta}-unsaturated carboxylic acids are abundant among anaerobic and facultatively anaerobic microorganisms, yet substrate specificity has been experimentally verified for few encoded proteins. Here, we co-produced in Escherichia coli one such protein of the marine bacterium Vibrio ruber (GenBank SJN56021; annotated as urocanate reductase) with Vibrio cholerae flavin transferase. The isolated protein (named Crd) is a heterodimer of the SJN56021-encoded subunit CrdB (NADH:flavin, FAD binding 2, and FMN bind domains) and an additional subunit CrdA (SJN56019, a single NADH:flavin domain) that interact via their NADH:flavin domains (Alphafold2 prediction). Each domain contains a flavin group (three FMNs and one FAD in total), one of the FMN groups being linked covalently by the flavin transferase. Crd readily reduces cinnamate, p-coumarate, caffeate, and ferulate under anaerobic conditions with NADH or methyl viologen as the electron donor, is moderately active against acrylate and practically inactive against urocanate. The reduction reactions started by NADH demonstrated a time lag of several minutes, suggesting a redox regulation of Crd activity. The oxidized enzyme is inactive, which apparently prevents production of reactive oxygen species under aerobic conditions. Our findings identify Crd as a regulated NADH-dependent cinnamate reductase, apparently protecting V. ruber from cinnamate poisoning. Abbreviated SummaryThe genome of the marine bacterium Vibrio ruber encodes a heterodimeric NADH-dependent cinnamate reductase, apparently protecting V. ruber from poisoning by cinnamate and its derivatives. The reductase contains four flavin groups, one being linked covalently, and appears to be redox-regulated. Oxidized enzyme is inactive, which apparently prevents production of reactive oxygen species under aerobic conditions.