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 Konas, Kulwant Aulak, Michael Regulski, Tim Tully, Dennis J. Stuehr, Sanjay Ghosh

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7 Citations (Scopus)

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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Drosophila melanogaster
Nitric Oxide Synthase
Oxidation-Reduction
Oxidoreductases
Calmodulin
Fruits
Catalysis
NADP
Diptera
Fruit
Electrons
Cytochrome Reductases
Flavin Mononucleotide
Flavin-Adenine Dinucleotide
Enzymes
Cytochromes c
Shielding
Charge transfer
Conservation
Binding Sites

Cite this

Ray, Sougata Sinha ; Sengupta, Rajib ; Tiso, Mauro ; Haque, Mohammad Mahfuzul ; Sahoo, Rupam ; Konas, David ; Aulak, Kulwant ; Regulski, Michael ; Tully, Tim ; Stuehr, Dennis J. ; Ghosh, Sanjay. / Reductase domain of Drosophila melanogaster nitric-oxide synthase : Redox transformations, regulation, and similarity to mammalian homologues. In: Biochemistry. 2007 ; Vol. 46, No. 42. pp. 11865-11873.
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title = "Reductase domain of Drosophila melanogaster nitric-oxide synthase: Redox transformations, regulation, and similarity to mammalian homologues",
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.",
author = "Ray, {Sougata Sinha} and Rajib Sengupta and Mauro Tiso and Haque, {Mohammad Mahfuzul} and Rupam Sahoo and David Konas and Kulwant Aulak and Michael Regulski and Tim Tully and Stuehr, {Dennis J.} and Sanjay Ghosh",
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Ray, SS, Sengupta, R, Tiso, M, Haque, MM, Sahoo, R, Konas, D, Aulak, K, Regulski, M, Tully, T, Stuehr, DJ & Ghosh, S 2007, 'Reductase domain of Drosophila melanogaster nitric-oxide synthase: Redox transformations, regulation, and similarity to mammalian homologues', Biochemistry, vol. 46, no. 42, pp. 11865-11873. https://doi.org/10.1021/bi700805x

Reductase domain of Drosophila melanogaster nitric-oxide synthase : Redox transformations, regulation, and similarity to mammalian homologues. / Ray, Sougata Sinha; Sengupta, Rajib; Tiso, Mauro; Haque, Mohammad Mahfuzul; Sahoo, Rupam; Konas, David; Aulak, Kulwant; Regulski, Michael; Tully, Tim; Stuehr, Dennis J.; Ghosh, Sanjay.

In: Biochemistry, Vol. 46, No. 42, 23.10.2007, p. 11865-11873.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Reductase domain of Drosophila melanogaster nitric-oxide synthase

T2 - Redox transformations, regulation, and similarity to mammalian homologues

AU - Ray, Sougata Sinha

AU - Sengupta, Rajib

AU - Tiso, Mauro

AU - Haque, Mohammad Mahfuzul

AU - Sahoo, Rupam

AU - Konas, David

AU - Aulak, Kulwant

AU - Regulski, Michael

AU - Tully, Tim

AU - Stuehr, Dennis J.

AU - Ghosh, Sanjay

PY - 2007/10/23

Y1 - 2007/10/23

N2 - 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.

AB - 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.

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U2 - 10.1021/bi700805x

DO - 10.1021/bi700805x

M3 - Article

VL - 46

SP - 11865

EP - 11873

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 42

ER -