Relationship between structure and function of the NADH oxidase from Lactobacillus brevis.

Water-producing NADH oxidases catalyse the oxidation of NADH by molecular oxygen to generate NAD+ and water. These enzymes require FAD as cofactor to carry out their enzymatic activity and contribute to bacterial protection against oxidative stress. They have received considerable attention since their NAD+ recycling activity could make them candidates of choice for various industrial oxidoreductive processes. However, most of these enzymes are produced in recombinant hosts (e.g., E. coli) as apoenzymes and therefore require activation by incubation with FAD. In this study, we describe the characterization of the NADH oxidase from Lactobacillus brevis (LbNOX), a homodimeric flavoenzyme containing one non-covalently bound FAD molecule per monomer. In this paper, we show that the production, purification and formulation of LbNOX result in a heterogeneous enzyme solution. The active dimeric form is pH dependent and correlates with the presence of FAD. We also performed a comprehensive bioinformatic analysis of the LbNOX structure, which highlights crucial residues for pH-dependent dimerisation and shows that FAD is tightly bound at the dimerisation interface. This structural and functional characterization is crucial for a complete understanding of the enzyme's activation mechanism and will support the development of a robust and reproducible protocol for the production, purification and formulation of a fully active and homogeneous enzyme solution. More broadly, this work will contribute to the development of NADH oxidases based industrial applications and their FAD-dependent activation mechanism.