title: Identification of redox partners and development of a novel chimeric bacterial nitric oxide synthase for structure activity analyses.
abstract: Production of nitric oxide (NO) by nitric oxide synthase (NOS) requires electrons to reduce the heme iron for substrate oxidation. Both FAD and FMN flavin groups mediate the transfer of NADPH derived electrons to NOS. Unlike mammalian NOS that contain both FAD and FMN binding domains within a single polypeptide chain, bacterial NOS is only composed of an oxygenase domain and must rely on separate redox partners for electron transfer and subsequent activity. Here, we report on the native redox partners for Bacillus subtilis NOS (bsNOS) and a novel chimera that promotes bsNOS activity. By identifying and characterizing native redox partners, we were also able to establish a robust enzyme assay for measuring bsNOS activity and inhibition. This assay was used to evaluate a series of established NOS inhibitors. Using the new assay for screening small molecules led to the identification of several potent inhibitors for which bsNOS-inhibitor crystal structures were determined. In addition to characterizing potent bsNOS inhibitors, substrate binding was also analyzed using isothermal titration calorimetry giving the first detailed thermodynamic analysis of substrate binding to NOS.
authors.affiliation: From the Departments of Molecular Biology and Biochemistry.
authors.forename: Jeffrey K
authors.initials: JK
authors.lastname: Holden
authors.affiliation: Pharmaceutical Sciences, and.
authors.forename: Nathan
authors.initials: N
authors.lastname: Lim
authors.affiliation: From the Departments of Molecular Biology and Biochemistry, Pharmaceutical Sciences, and Chemistry, University of California, Irvine, California 92697-3900 poulos@uci.edu.
authors.forename: Thomas L
authors.initials: TL
authors.lastname: Poulos
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry*; D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000235:genetics; D002151:Calorimetry; D003001:Cloning, Molecular; D045304:Cytochromes c / Q000378:metabolism; D004583:Electrons; D004926:Escherichia coli / Q000378:metabolism; D005287:Ferredoxin-NADP Reductase / Q000235:genetics; D005418:Flavodoxin / Q000235:genetics; D006801:Humans; D007093:Imidazoles / Q000737:chemistry; D020128:Inhibitory Concentration 50; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D009573:Nitrites / Q000378:metabolism; D010084:Oxidation-Reduction; D051381:Rats
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D005418:Flavodoxin; D007093:Imidazoles; D009573:Nitrites; D009569:Nitric Oxide; D009249:NADP; C029899:imidazole; D045304:Cytochromes c; D019001:Nitric Oxide Synthase; D005287:Ferredoxin-NADP Reductase; D009247:NADH, NADPH Oxidoreductases; C116041:flavodoxin NADPH oxidoreductase
keywords: Crystallography; Electron Transfer; Enzyme Inhibitor; Isothermal Titration Calorimetry (ITC); Nitric Oxide