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 ϩ ) 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 ϩ from these decomposition products; however, the importance of the salvage pathway for survival is not well elucidated, except for in pathogens lacking the NAD ϩ de novo synthesis pathway. Herein, we report the importance of the NAD ϩ 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 ϩ salvage pathway. This recombinant enzyme has a high catalytic activity against nicotinamide (K m of 17 M, k cat of 50 s Ϫ1 , k cat /K m of 3.0 ϫ 10 3 s Ϫ1 · mM Ϫ1 ). Deletion of this gene abolished nicotinamide deamination activity in crude extracts of T. thermophilus and disrupted the NAD ϩ 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 ϩ and NADH.IMPORTANCE NAD ϩ 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 ϩ decomposition is, in general, more rapid. This study emphasizes that NAD ϩ instability and its homeostasis can be one of the important factors for thermophile survival in extreme temperatures.KEYWORDS NAD ϩ , Thermus thermophilus, nicotinamidase, salvage synthesis N AD (NAD ϩ ) is an essential molecule for cellular metabolism. It serves as an electron donor/acceptor for many redox reactions in cellular metabolism. It also serves as a precursor for NADP ϩ and a substrate for both bacterial DNA ligases and ADP ribosyl transferases (1, 2). Due to its importance as a cofactor for many oxidoreductases, NAD ϩ is also an essential compound in biotechnology applications (3); however, NAD ϩ is known to be chemically unstable, especially at high temperatures, where it nonenzymatically decomposes to ADP-ribose and nicotinamide (4,5).Cells need to maintain a certain concentration of NAD ϩ for metabolism, and different organisms possess different pathways for synthesizing NAD ϩ , such as the de novo biosynthesis pathway and the salvage pathway (6). In the salvage pathway,

Hyperthermophilic Archaeon Thermococcus kodakarensis Utilizes a Four-Step Pathway for NAD + Salvage through Nicotinamide Deamination

“…Additionally, it is well known that NAD ϩ is a thermolabile molecule (10,11) and that its degradation results in the generation of ADP-ribose and Nm (12,13). This is particularly relevant in thermophilic organisms, and several studies have examined the mechanisms that are involved in regenerating NAD ϩ from its degradation products (13,14).…”

Response surface optimization of conditions for culturing <i>Azotobacter chroococcum</i> in <i>Agaricus bisporus</i> industrial wastewater

“…The temperature influences microbial life mainly by affecting the mobility of the microbial cell membrane and the activity of biological macromolecules. As the temperature increases, the rates of intracellular enzymatic reactions increase, resulting in an increase in cell metabolism and growth (Taniguchi et al, 2017). However, once the temperature becomes too high, the bioactive substances become denatured, resulting in decreased cell functions and even death.…”

Developing a single strain for in vitro salvage synthesis of NAD+ at high temperatures and its potential for bioconversion

“…By screening or designing enzymes with better performance, the performance of in vitro pathway can be accumulatively improved. Salvage synthesis of NAD + is widely distributed among thermophilic bacteria as well as hyperthermophilic archaea as a means to address thermal instability of NAD + [30, 31]. A diverse compendium of enzymes is available for consideration for inclusion in a NAD + salvage synthesis system.…”