The reductase domain of the human inducible nitric oxide synthase is fully active in the absence of bound calmodulin.
Nitric oxide synthases (NOSs) are composed of a flavin-containing reductase domain and a heme-containing oxygenase domain. Each NOS enzyme also contains a calmodulin (CaM) binding domain and requires bound calmodulin for enzymatic activity. The CaM binding properties of the different NOS isozymes differ in the need for free calcium ions (Ca2+). We investigated CaM binding using reductase domains from the human and mouse inducible as well as the rat neuronal isoforms of NOS. An Escherichia coli expression system was designed to generate truncated recombinant NOS proteins for each isoform in which an extended CaM binding domain was either included or deleted. The reductase domains with the extended N-terminal CaM binding domains of human iNOS (residues 480-1153) and mouse iNOS (residues 474-1144) show Ca2+ binding properties that are similar to their respective holoenzymes. In addition, the iNOS reductases were active in the presence or absence of CaM. Thus, CaM does not stimulate NADPH utilization of the reductase domain in iNOS enzymes. In contrast, the rat nNOS reductase enzymes showed Ca2+-dependent CaM binding and activation.
Newton
D C
DC
Department of Chemistry and Guelph Waterloo Centre for Graduate Work in Chemistry and Biochemistry, University of Waterloo, Waterloo, Ontario, N2L 3G1, Canada.
Montgomery
H J
HJ
Guillemette
J G
JG
D000818:Animals; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism; D002384:Catalysis; D004789:Enzyme Activation / Q000235:genetics / Q000502:physiology; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000096:biosynthesis / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051381:Rats; D017384:Sequence Deletion; D013053:Spectrophotometry
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D007527:Isoenzymes; D010446:Peptide Fragments; D010088:Oxidoreductases; C496320:NOS1 protein, human; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; C496321:Nos1 protein, mouse; C496322:Nos1 protein, rat; C496317:Nos2 protein, mouse; C496318:Nos2 protein, rat