A key enzyme of the NAD⁺salvage pathway in Thermus thermophilus: Characterization of nicotinamidase and the impact of its gene deletion at high temperatures

Abstract

© 2017 American Society for Microbiology. All Rights Reserved. NAD (NAD+) is a cofactor related to many cellular processes. This cofactor is known to be unstable, especially at high temperatures, where it chemically decomposes to nicotinamide and ADP-ribose. Bacteria, yeast, and higher organisms possess the salvage pathway for reconstructing NAD+from these decomposition products; however, the importance of the salvage pathway for survival is not well elucidated, except for in pathogens lacking the NAD+de novo synthesis pathway. Herein, we report the importance of the NAD+salvage pathway in the thermophilic bacterium Thermus thermophilus HB8 at high temperatures. We identified the gene encoding nicotinamidase (TTHA0328), which catalyzes the first reaction of the NAD+salvage pathway. This recombinant enzyme has a high catalytic activity against nicotinamide (Kmof 17 μM, kcatof 50 s-1, kcat/Kmof 3.0 × 103 s-1· mM-1). Deletion of this gene abolished nicotinamide deamination activity in crude extracts of T. thermophilus and disrupted the NAD+salvage pathway in T. thermophilus. Disruption of the salvage pathway led to the severe growth retardation at a higher temperature (80°C), owing to the drastic decrease in the intracellular concentrations of NAD+and NADH.