RGD Reference Report - A conserved aspartate (Asp-1393) regulates NADPH reduction of neuronal nitric-oxide synthase: implications for catalysis. - Rat Genome Database

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A conserved aspartate (Asp-1393) regulates NADPH reduction of neuronal nitric-oxide synthase: implications for catalysis.

Authors: Panda, K  Adak, S  Konas, D  Sharma, M  Stuehr, DJ 
Citation: Panda K, etal., J Biol Chem 2004 Apr 30;279(18):18323-33. Epub 2004 Feb 13.
RGD ID: 1358520
Pubmed: PMID:14966111   (View Abstract at PubMed)
DOI: DOI:10.1074/jbc.M310391200   (Journal Full-text)

Nitric-oxide synthases (NOSs) are flavo-heme enzymes whose electron transfer reactions are controlled by calmodulin (CaM). The NOS flavoprotein domain includes a ferredoxin-NADP(+) reductase (FNR)-like module that contains NADPH- and FAD-binding sites. FNR-like modules in related flavoproteins have three conserved residues that regulate electron transfer between bound NAD(P)H and FAD. To investigate the function of one of these residues in neuronal NOS (nNOS), we generated and characterized mutants that had Val, Glu, or Asn substituted for the conserved Asp-1393. All three mutants exhibited normal composition, spectral properties, and binding of cofactors, substrates, and CaM. All had slower NADPH-dependent cytochrome c and ferricyanide reductase activities, which were associated with proportionally slower rates of NADPH-dependent flavin reduction in the CaM-free and CaM-bound states. Rates of NO synthesis were also proportionally slower in the mutants and were associated with slower rates of CaM-dependent ferric heme reduction. However, a D1393V mutant whose flavins had been prereduced with NADPH had a normal rate of heme reduction. This indicated that the kinetic defect was restricted to flavin reduction step(s) in the mutants and suggested that this limited their catalytic activities. Together, our results show the following. 1) The presence and positioning of the Asp-1393 carboxylate side chain are critical to enable NADPH-dependent reduction of the nNOS flavoprotein. 2) Control of flavin reduction is important because it ensures that the rate of heme reduction is sufficiently fast to enable NO synthesis by nNOS.

Gene Ontology Annotations    Click to see Annotation Detail View

Biological Process
TermQualifierEvidenceWithReferenceNotesSourceOriginal Reference(s)
nitric oxide biosynthetic process  IDA 1358520 RGD 

Molecular Function
TermQualifierEvidenceWithReferenceNotesSourceOriginal Reference(s)
calmodulin binding  IDA 1358520 RGD 
flavin adenine dinucleotide binding  IDA 1358520 RGD 
FMN binding  IDA 1358520 RGD 
NADPH binding  IDA 1358520 RGD 
nitric-oxide synthase activity  IDA 1358520 RGD 

Objects Annotated

Genes (Rattus norvegicus)
Nos1  (nitric oxide synthase 1)

Objects referenced in this article
Gene Nos2 nitric oxide synthase 2 Rattus norvegicus

Additional Information