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

• Published in: PloS one Vol. 12; no. 7; p. e0181561
• Main Authors: Zapata-Pérez, Rubén, Martínez-Moñino, Ana-Belén, García-Saura, Antonio-Ginés, Cabanes, Juana, Takami, Hideto, Sánchez-Ferrer, Álvaro
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 27.07.2017; Public Library of Science (PLoS)
• Subjects: Aldehydes - metabolism; Amino Acid Sequence; Analogs; Bacteria - enzymology; Binding sites; Biochemistry; Bioinformatics; Biology and Life Sciences; Biomedical research; Catalysis; Communities; Computer and Information Sciences; Deoxyribonucleic acid; DNA; Enzyme Inhibitors - pharmacology; Enzyme kinetics; Enzyme Stability - drug effects; Extreme environments; Homeostasis; Hot Springs - microbiology; Hydrogen-Ion Concentration; Hydrolases; Iron; Kinetics; Microbial mats; Microbiota; Microorganisms; Models, Molecular; Molecular biology; Mutagenesis; Mutant Proteins - metabolism; NAD; Nicotinamidase; Nicotinamidase - antagonists & inhibitors; Nicotinamidase - chemistry; Nicotinamidase - metabolism; Nicotinamide; Nicotinic acid; pH effects; Phylogeny; Physical Sciences; Physiological aspects; Properties; Protein kinase A; Proteins; Research and Analysis Methods; Rivers - microbiology; Sequence Alignment; Sequence Homology, Amino Acid; Subgroups; Substrate Specificity; Substrates; Temperature; Thermal stability; Tryptophan; Water Microbiology; Japan; Spain
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0181561

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.