A Redox-Regulated, Heterodimeric NADH:cinnamate Reductase in Vibrio ruber

AbstractGenes of putative reductases of α,β-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 a heterodimeric protein of the facultatively anaerobic marine bacteriumVibrio ruber (GenBank SJN56019 and SJN56021; annotated as NADPH azoreductase and urocanate reductase, respectively) withVibrio cholerae flavin transferase. The isolated protein (named Crd) consists of thesjn56021-encoded subunit CrdB (NADH:flavin,FAD binding  2, andFMN bind domains) and an additional subunit CrdA (SJN56019, a singleNADH:flavin domain) that interact via theirNADH: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 and fumarate. Cinnamates induced Crd synthesis inV.  ruber cells grown aerobically or anaerobically. The Crd-catalyzed reduction started by NADH demonstrated a time lag of several minutes, suggesting a redox regulation of the enzyme activity. The oxidized enzyme is inactive, which apparently prevents production of reactive oxygen species under aerobic conditi...
Source: Biochemistry (Moscow) - Category: Biochemistry Source Type: research