A novel, NADH-dependent acrylate reductase in Vibrio harveyi

Abstract

Bacteria coping with oxygen deficiency use alternative terminal electron acceptors for NADH regeneration, particularly fumarate. Fumarate is reduced by the FAD_binding_2 domain of cytoplasmic fumarate reductase in many bacteria. The variability of the primary structure of this domain in homologous proteins suggests the existence of reducing activities with different specificities. Here we produced and characterized one such protein, Vibrio harveyi ARD, and found it to be a specific NADH:acrylate oxidoreductase. This previously unknown enzyme contains covalently bound FMN and non-covalently bound FAD and FMN in a ratio of 1:1:1. The covalently bound FMN is absolutely required for activity and is attached by the specific flavin transferase, ApbE, to a threonine residue in the auxiliary FMN_bind domain. RT-qPCR and activity measurements indicated dramatic stimulation of ARD biosynthesis by acrylate in the V. harveyi cells grown aerobically. In contrast, the ard gene expression in the cells grown anaerobically was high without acrylate and increased only twofold in its presence. These findings suggest that the principal role of ARD in Vibrio is energy-saving detoxification of acrylate coming from the environment. ImportanceThe benefits of the massive genomic information accumulated in recent years for biological sciences have been limited by the lack of data on the function of most gene products. Approximately half of the known prokaryotic genes are annotated as "proteins with unknown functions," and many other genes are annotated incorrectly. Thus, the functional and structural characterization of the products of such genes, including identification of all existing enzymatic activities, is a pressing issue in modern biochemistry. In this work, we have shown that the ard gene product of V. harveyi (GenBank ID: AIV07243) exhibits a yet undescribed NADH:acrylate oxidoreductase activity. This activity may allow acrylate detoxification and its use as a terminal electron acceptor in anaerobic or substrate in aerobic respiration of marine and other bacteria.