Taniguchi Hironori, Sungwallek Sathidaphorn, Chotchuang Phatcharin, Okano Kenji, Honda Kohsuke
Synthetic Bioengineering Lab, Department of Biotechnology, Graduate School of Engineering, Osaka University, Osaka, Japan.
Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok, Thailand.
J Bacteriol. 2017 Aug 8;199(17). doi: 10.1128/JB.00359-17. Print 2017 Sep 1.
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 synthesis pathway. Herein, we report the importance of the NAD salvage pathway in the thermophilic bacterium 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 ( of 17 μM, of 50 s, / of 3.0 × 10 s · mM). Deletion of this gene abolished nicotinamide deamination activity in crude extracts of and disrupted the NAD salvage pathway in 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. 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.
烟酰胺腺嘌呤二核苷酸（NAD）是一种与许多细胞过程相关的辅助因子。已知这种辅助因子不稳定，尤其是在高温下，它会化学分解为烟酰胺和ADP - 核糖。细菌、酵母和高等生物拥有从这些分解产物中重建NAD的补救途径；然而，除了缺乏NAD合成途径的病原体外，补救途径对生存的重要性尚未得到充分阐明。在此，我们报告了嗜热细菌HB8中NAD补救途径在高温下的重要性。我们鉴定出编码烟酰胺酶（TTHA0328）的基因，该酶催化NAD补救途径的第一步反应。这种重组酶对烟酰胺具有高催化活性（Km为17μM，kcat为50 s-1，kcat/Km为3.0×104 s-1·mM-1）。该基因的缺失消除了HB8粗提物中的烟酰胺脱氨活性，并破坏了HB8中的NAD补救途径。补救途径的破坏导致在较高温度（80°C）下严重的生长迟缓，这是由于细胞内NAD和NADH浓度急剧下降所致。NAD和其他烟酰胺辅助因子对细胞代谢至关重要。这些分子不稳定，即使在大多数生物体的生理条件下也会分解。嗜热菌能够在高温下生存，而在高温下NAD的分解通常更快。这项研究强调，NAD的不稳定性及其稳态可能是嗜热菌在极端温度下生存的重要因素之一。
