Identification of a flavin mononucleotide module residue critical for activity of inducible nitrite oxide synthase.
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.
Liu
Xian-De
XD
Department of Medicine, Baylor College of Medicine, Houston, TX 77030, USA.
Mazumdar
Tuhina
T
Xu
Yi
Y
Getzoff
Elizabeth D
ED
Eissa
N Tony
NT
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
D001224:Aspartic Acid; D009569:Nitric Oxide; D018698:Glutamic Acid; D005486:Flavin Mononucleotide; D052247:Nitric Oxide Synthase Type II