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

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 (K_m of 17 μM, k_cat of 50 s⁻¹, k_cat/K_m of 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.

IMPORTANCE NAD⁺ and other nicotinamide cofactors are essential for cell metabolism. These molecules are unstable and decompose, even under the physiological conditions in most organisms. Thermophiles can survive at high temperatures where NAD⁺ decomposition is, in general, more rapid. This study emphasizes that NAD⁺ instability and its homeostasis can be one of the important factors for thermophile survival in extreme temperatures.