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

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Yulia V. Bertsova^1,a, Marina V. Serebryakova^1,b, Victor A. Anashkin^1,c, Alexander A. Baykov^1,d, and Alexander V. Bogachev^1,e*

¹Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119234 Moscow, Russia

Received June 24, 2023; Revised October 9, 2023; Accepted October 10, 2023

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Genes 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 bacterium Vibrio ruber (GenBank SJN56019 and SJN56021; annotated as NADPH azoreductase and urocanate reductase, respectively) with Vibrio cholerae flavin transferase. The isolated protein (named Crd) consists 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 and fumarate. Cinnamates induced Crd synthesis in V. 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 conditions. Our findings identify Crd as a regulated NADH-dependent cinnamate reductase, apparently protecting V. ruber from (hydroxy)cinnamate poisoning.

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KEY WORDS: anaerobic respiration, cinnamic acid, caffeic acid, enzyme regulation, reactive oxygen species, rhizosphere, Vibrio

DOI: 10.1134/S0006297924020056

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