Reductase Domain of Drosophila melanogaster Nitric-Oxide Synthase: Redox Transformations, Regulation, and Similarity to Mammalian Homologues

Ray, S. S., Sengupta, R., Tiso, M., Haque, M. M., Sahoo, R., Konas, D. W., Aulak, K., Regulski, M. R., 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). pp. 11865-11873. ISSN 0006-2960 (Print)

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URL: http://pubs.acs.org/cgi-bin/article.cgi/bichaw/200...
DOI: 10.1021/bi700805x S0006-2960(70)00805-X

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 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.

Item Type: Paper
Uncontrolled Keywords: MACROPHAGE NO SYNTHASE ELECTRON-TRANSFER CYCLIC-GMP Isolated Oxygenase FLAVIN DOMAIN CALMODULIN EXPRESSION CATALYSIS enzyme IDENTIFICATION
Subjects: organism description > animal > mammal
organs, tissues, organelles, cell types and functions > cell types and functions > cell types > neurons
organs, tissues, organelles, cell types and functions > cell types and functions > cell types > neurons
organs, tissues, organelles, cell types and functions > cell types and functions > cell types > neurons

bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > enzymes > nitric oxide synthase
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein expression
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Depositing User: CSHL Librarian
Date Deposited: 03 Nov 2011 15:24
Last Modified: 29 Nov 2011 20:08
URI: http://repository.cshl.edu/id/eprint/23126

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