Reductase domain of Drosophila melanogaster nitric-oxide synthase: Redox transformations, regulation, and similarity to mammalian homologues

Sougata Sinha Ray, Rajib Sengupta, Mauro Tiso, Mohammad Mahfuzul Haque, Rupam Sahoo, David W. Konas, Kulwant Aulak, Michael Regulski, Tim Tully, Dennis J. Stuehr, Sanjay Ghosh

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Abstract

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 Ca 2+ 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.

Original languageEnglish
Pages (from-to)11865-11873
Number of pages9
JournalBiochemistry
Volume46
Issue number42
DOIs
StatePublished - 23 Oct 2007

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Ray, S. S., Sengupta, R., Tiso, M., Haque, M. M., Sahoo, R., Konas, D. W., Aulak, K., Regulski, M., Tully, T., Stuehr, D. J., & Ghosh, S. (2007). Reductase domain of Drosophila melanogaster nitric-oxide synthase: Redox transformations, regulation, and similarity to mammalian homologues. Biochemistry, 46(42), 11865-11873. https://doi.org/10.1021/bi700805x