Biochemical characterization of a new nicotinamidase from an unclassified bacterium thriving in a geothermal water stream microbial mat community

Nicotinamidases are amidohydrolases that convert nicotinamide into nicotinic acid, contributing to NAD+ homeostasis in most organisms. In order to increase the number of nicotinamidases described to date, this manuscript characterizes a nicotinamidase obtained from a metagenomic library fosmid clone (JFF054_F02) obtained from a geothermal water stream microbial mat community in a Japanese epithermal mine. The enzyme showed an optimum temperature of 90°C, making it the first hyperthermophilic bacterial nicotinamidase to be characterized, since the phylogenetic analysis of this fosmid clone placed it in a clade of uncultured geothermal bacteria. The enzyme, named as UbNic, not only showed an alkaline optimum pH, but also a biphasic pH dependence of its kcat, with a maximum at pH 9.5–10.0. The two pKa values obtained were 4.2 and 8.6 for pKes1 and pKes2, respectively. These results suggest a possible flexible catalytic mechanism for nicotinamidases, which reconciles the two previously proposed mechanisms. In addition, the enzyme showed a high catalytic efficiency, not only toward nicotinamide, but also toward other nicotinamide analogs. Its mutational analysis showed that a tryptophan (W83) is needed in one of the faces of the active site to maintain low Km values toward all the substrates tested. Furthermore, UbNic proved to contain a Fe2+ ion in its metal binding site, and was revealed to belong to a new nicotinamidase subgroup. All these characteristics, together with its high pH- and thermal stability, distinguish UbNic from previously described nicotinamidases, and suggest that a wide diversity of enzymes remains to be discovered in extreme environments.

烟酰胺酶（nicotinamidases）是一类可将烟酰胺转化为烟酸的酰胺水解酶（amidohydrolases），在绝大多数生物中参与维持烟酰胺腺嘌呤二核苷酸（NAD+）的稳态。为扩充迄今已报道的烟酰胺酶数量，本研究对一株源自日本浅成热液矿地热溪流微生物垫群落的宏基因组文库（metagenomic library）黏粒克隆（fosmid clone，JFF054_F02）所编码的烟酰胺酶进行了表征。该酶的最适温度为90℃，是首个被表征的超嗜热细菌源烟酰胺酶——该黏粒克隆的系统发育分析（phylogenetic analysis）结果显示，其隶属于未培养地热细菌的进化枝（clade）。该酶被命名为UbNic，不仅具有碱性最适pH，其催化常数（kcat）还呈现双相pH依赖性，在pH 9.5~10.0时达到峰值。测得的两个解离常数（pKa）分别为pKes1=4.2与pKes2=8.6。上述结果表明烟酰胺酶可能存在灵活的催化机制，可调和此前提出的两种催化机制假说。此外，该酶不仅对烟酰胺具有较高催化效率，对其他烟酰胺类似物亦表现出优异的催化效能。突变分析显示，活性位点（active site）一侧的色氨酸残基（W83）是维持所有受试底物低米氏常数（Km）的关键残基。进一步研究证实，UbNic的金属结合位点（metal binding site）含有Fe²+离子，且其隶属于一个全新的烟酰胺酶亚群。上述特性，加之其优异的酸碱与热稳定性，使UbNic区别于此前已报道的所有烟酰胺酶，同时也提示极端环境中仍存在大量待发掘的酶类多样性。