title: Identification of a flavin mononucleotide module residue critical for activity of inducible nitrite oxide synthase.
abstract: Inducible NO synthase (iNOS) contains an amino-terminal oxygenase domain, a carboxy-terminal reductase domain, and an intervening calmodulin-binding domain. For the synthesis of NO, iNOS is active as a homodimer formed by oxygenase domains, while the reductase domain is required to transfer electrons from NADPH. In this study, we identify glutamate 658 in the FMN domain of human iNOS to be a critical residue for iNOS activity and we explore the underlying mechanism for such role. Mutation of glutamate to aspartate almost abolished iNOS activity and reduced dimer formation. Substitution of this residue with noncharged alanine and glutamine, or positively charged lysine did not affect dimer formation and maintained around 60% of iNOS activity. These results suggest that the negative charge specific to glutamate plays an important role in iNOS activity.
authors.affiliation: Department of Medicine, Baylor College of Medicine, Houston, TX 77030, USA.
authors.forename: Xian-De
authors.initials: XD
authors.lastname: Liu
authors.forename: Tuhina
authors.initials: T
authors.lastname: Mazumdar
authors.forename: Yi
authors.initials: Y
authors.lastname: Xu
authors.forename: Elizabeth D
authors.initials: ED
authors.lastname: Getzoff
authors.forename: N Tony
authors.initials: NT
authors.lastname: Eissa
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics; D002460:Cell Line; D019281:Dimerization; D004789:Enzyme Activation / Q000235:genetics; D005486:Flavin Mononucleotide / Q000737:chemistry / Q000502:physiology*; D018698:Glutamic Acid / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009569:Nitric Oxide / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
chemical_list: D001224:Aspartic Acid; D009569:Nitric Oxide; D018698:Glutamic Acid; D005486:Flavin Mononucleotide; D052247:Nitric Oxide Synthase Type II