Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto, Japan.
JST, CREST, Chiyoda-ku, Tokyo, Japan.
J Bacteriol. 2018 May 9;200(11). doi: 10.1128/JB.00785-17. Print 2018 Jun 1.
Many organisms possess pathways that regenerate NAD from its degradation products, and two pathways are known to salvage NAD from nicotinamide (Nm). One is a four-step pathway that proceeds through deamination of Nm to nicotinic acid (Na) by Nm deamidase and phosphoribosylation to nicotinic acid mononucleotide (NaMN), followed by adenylylation and amidation. Another is a two-step pathway that does not involve deamination and directly proceeds with the phosphoribosylation of Nm to nicotinamide mononucleotide (NMN), followed by adenylylation. Judging from genome sequence data, the hyperthermophilic archaeon is supposed to utilize the four-step pathway, but the fact that the adenylyltransferase encoded by TK0067 recognizes both NMN and NaMN also raises the possibility of a two-step salvage mechanism. Here, we examined the substrate specificity of the recombinant TK1676 protein, annotated as nicotinic acid phosphoribosyltransferase. The TK1676 protein displayed significant activity toward Na and phosphoribosyl pyrophosphate (PRPP) and only trace activity with Nm and PRPP. We further performed genetic analyses on TK0218 (quinolinic acid phosphoribosyltransferase) and TK1650 (Nm deamidase), involved in biosynthesis and four-step salvage of NAD, respectively. The ΔTK0218 mutant cells displayed growth defects in a minimal synthetic medium, but growth was fully restored with the addition of Na or Nm. The ΔTK0218 ΔTK1650 mutant cells did not display growth in the minimal medium, and growth was restored with the addition of Na but not Nm. The enzymatic and genetic analyses strongly suggest that NAD salvage in requires deamination of Nm and proceeds through the four-step pathway. Hyperthermophiles must constantly deal with increased degradation rates of their biomolecules due to their high growth temperatures. Here, we identified the pathway that regenerates NAD from nicotinamide (Nm) in the hyperthermophilic archaeon The organism utilizes a four-step pathway that initially hydrolyzes the amide bond of Nm to generate nicotinic acid (Na), followed by phosphoribosylation, adenylylation, and amidation. Although the two-step pathway, consisting of only phosphoribosylation of Nm and adenylylation, seems to be more efficient, Nm mononucleotide in the two-step pathway is much more thermolabile than Na mononucleotide, the corresponding intermediate in the four-step pathway. Although NAD itself is thermolabile, this may represent an example of a metabolism that has evolved to avoid the use of thermolabile intermediates.
许多生物体拥有从 NAD 的降解产物中再生 NAD 的途径，已知有两种途径可以从烟酰胺 (Nm) 中回收 NAD。一种是四步途径，通过 Nm 脱氨酶将 Nm 脱氨为烟酸 (Na)，然后通过磷酸核糖基化转化为烟酸单核苷酸 (NaMN)，接着进行腺苷酰化和酰胺化。另一种是两步途径，不涉及脱氨，直接通过 Nm 的磷酸核糖基化生成烟酰胺单核苷酸 (NMN)，然后进行腺苷酰化。根据基因组序列数据，推测嗜热古菌 利用四步途径，但 TK0067 编码的腺苷酰基转移酶既能识别 NMN 又能识别 NaMN，这也增加了两步回收机制的可能性。在这里，我们研究了 TK1676 蛋白的重组体（注释为烟酸磷酸核糖基转移酶）的底物特异性。TK1676 蛋白对 Na 和磷酸核糖基焦磷酸 (PRPP) 表现出显著的活性，而对 Nm 和 PRPP 仅表现出微量的活性。我们进一步对参与 NAD 生物合成和四步回收的 TK0218（喹啉酸磷酸核糖基转移酶）和 TK1650（Nm 脱氨酶）进行了遗传分析。ΔTK0218 突变细胞在最小合成培养基中表现出生长缺陷，但添加 Na 或 Nm 可完全恢复生长。ΔTK0218 ΔTK1650 突变细胞在最小培养基中无法生长，添加 Na 可恢复生长，但添加 Nm 则不可。酶学和遗传学分析强烈表明， 在 中 NAD 的回收需要 Nm 的脱氨作用，并通过四步途径进行。嗜热微生物由于其较高的生长温度，必须不断应对其生物分子降解速率的增加。在这里，我们鉴定了在嗜热古菌 中从烟酰胺 (Nm) 再生 NAD 的途径。该生物体利用四步途径，首先水解 Nm 的酰胺键生成烟酸 (Na)，然后进行磷酸核糖基化、腺苷酰化和酰胺化。尽管两步途径仅包括 Nm 的磷酸核糖基化和腺苷酰化，但似乎更有效率，但两步途径中的 Nm 单核苷酸比四步途径中的相应中间体 Na 单核苷酸更不稳定。尽管 NAD 本身不稳定，但这可能代表了一种为避免使用不稳定中间体而进化的代谢。
