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

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• 作者：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
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：October 23, 2007
• 年：2007
• 卷：46
• 期：42
• 页码：11865 - 11873
• 全文大小：195K
• 年卷期：v.46,no.42(October 23, 2007)
• ISSN：1520-4995

文摘

The nitric oxide synthase of Drosophila melanogaster (dNOS) participates in essentialdevelopmental and behavioral aspects of the fruit fly, but little is known about dNOS catalysis andregulation. To address this, we expressed a construct comprising the dNOS reductase domain and itsadjacent 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 betweenthat of the mammalian endothelial and neuronal NOS enzymes. CaM binding caused the cytochrome creductase activity of dNOSr to increase 4 times and achieve an activity comparable to that of mammalianneuronal NOS. This change was associated with decreased shielding of the FMN cofactor from solventand an increase in the rate of NADPH-dependent flavin reduction. Flavin reduction in dNOSr was relativelyslow following the initial 2-electron reduction, suggesting a slow inter-flavin electron transfer, and nocharge-transfer complex was observed between bound NADP⁺ and reduced FAD during the process. Weconclude that dNOSr catalysis and regulation is most similar to the mammalian neuronal NOS reductasedomain, although differences exist in their flavin reduction behaviors. The apparent conservation betweenthe fruit fly and mammalian enzymes is consistent with dNOS operating in various signal cascades thatinvolve NO.