Reductase domain of Drosophila melanogaster nitric-oxide synthase: redox transformations, regulation, and similarity to mammalian homologues.
The nitric oxide synthase of Drosophila melanogaster (dNOS) participates in essential developmental and behavioral aspects of the fruit fly, but little is known about dNOS catalysis and regulation. To address this, we expressed a construct comprising the dNOS reductase domain and its adjacent calmodulin (CaM) binding site (dNOSr) and characterized the protein regarding its catalytic, kinetic, and regulatory properties. The Ca2+ concentration required for CaM binding to dNOSr was between that of the mammalian endothelial and neuronal NOS enzymes. CaM binding caused the cytochrome c reductase activity of dNOSr to increase 4 times and achieve an activity comparable to that of mammalian neuronal NOS. This change was associated with decreased shielding of the FMN cofactor from solvent and an increase in the rate of NADPH-dependent flavin reduction. Flavin reduction in dNOSr was relatively slow following the initial 2-electron reduction, suggesting a slow inter-flavin electron transfer, and no charge-transfer complex was observed between bound NADP+ and reduced FAD during the process. We conclude that dNOSr catalysis and regulation is most similar to the mammalian neuronal NOS reductase domain, although differences exist in their flavin reduction behaviors. The apparent conservation between the fruit fly and mammalian enzymes is consistent with dNOS operating in various signal cascades that involve NO.
Ray
Sougata Sinha
SS
Department of Biochemistry, University College of Sciences, Calcutta University, West Bengal 700 019, India.
Sengupta
Rajib
R
Tiso
Mauro
M
Haque
Mohammad Mahfuzul
MM
Sahoo
Rupam
R
Konas
David W
DW
Aulak
Kulwant
K
Regulski
Michael
M
Tully
Tim
T
Stuehr
Dennis J
DJ
Ghosh
Sanjay
S
D000818:Animals; D001665:Binding Sites; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D004331:Drosophila melanogaster / Q000201:enzymology*; D004579:Electron Transport; D004727:Endothelium / Q000201:enzymology; D005415:Flavins / Q000737:chemistry; D005420:Flavoproteins / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry*; D009474:Neurons / Q000201:enzymology* / Q000382:microbiology; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D010084:Oxidation-Reduction; D010802:Phylogeny; D011485:Protein Binding; D017434:Protein Structure, Tertiary
D002147:Calmodulin; D005415:Flavins; D005420:Flavoproteins; D009249:NADP; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D009251:NADPH-Ferrihemoprotein Reductase