Nitric oxide synthases (NOSs) are classified functionally, based on whether calmodulin binding is Ca 2؉ -dependent (cNOS) or Ca 2؉ -independent (iNOS). This key dichotomy has not been defined at the molecular level. Here we show that cNOS isoforms contain a unique polypeptide insert in their FMN binding domains which is not shared with iNOS or other related flavoproteins. Previously identified autoinhibitory domains in calmodulin-regulated enzymes raise the possibility that the polypeptide insert is the autoinhibitory domain of cNOSs. Consistent with this possibility, three-dimensional molecular modeling suggested that the insert originates from a site immediately adjacent to the calmodulin binding sequence. Synthetic peptides derived from the 45-amino acid insert of endothelial NOS were found to potently inhibit binding of calmodulin and activation of cNOS isoforms. This inhibition was associated with peptide binding to NOS, rather than free calmodulin, and inhibition could be reversed by increasing calmodulin concentration. In contrast, insert-derived peptides did not interfere with the arginine site of cNOS, as assessed from [ 3 H]N G -nitro-L-arginine binding, nor did they potently effect iNOS activity. Limited proteolysis studies showed that calmodulin's ability to gate electron flow through cNOSs is associated with displacement of the insert polypeptide; this is the first specific calmodulin-induced change in NOS conformation to be identified. Together, our findings strongly suggest that the insert is an autoinhibitory control element, docking with a site on cNOSs which impedes calmodulin binding and enzymatic activation. The autoinhibitory control element molecularly defines cNOSs and offers a unique target for developing novel NOS activators and inhibitors.Nitric oxide is a ubiquitous cell-signaling molecule, with protean roles in physiology and pathophysiology (1-3). Encoded by distinct genes, mammalian NO synthases (NOSs) 1 comprise a family of three calmodulin-dependent biopterohemoflavoproteins that are functionally distinguished by their modes of regulation (4). The two constitutively expressed isoforms of NOS (cNOSs), first identified in neuronal cells (nNOS) and endothelial cells (eNOS), remain dormant until calcium/calmodulin (Ca 2ϩ /CaM) binding is actuated by transient elevations in intracellular Ca 2ϩ . This Ca 2ϩ -dependent mode of regulation provides pulses of NO for moment-to-moment modulation of vascular tone and neurosignaling. In contrast, activity of the immunostimulant-induced isoform of NOS (iNOS) is Ca 2ϩ -independent, providing continuous high output NO generation for host defense. A remarkably high affinity for CaM, even at basally low levels of intracellular calcium, is responsible for the Ca 2ϩ independence of iNOS (5). Whether a given NOS isoform binds CaM in a Ca 2ϩ -dependent or -independent manner has been assumed to be a property solely of the amino acid sequence specified by a 20 -25-amino acid CaM binding site. However, this restrictive view is challenged by findings that ...

The sequences of nitric-oxide synthase flavin domains closely resemble that of NADPH-cytochrome P450 reductase (CPR). However, all nitric-oxide synthase (NOS) isoforms are 20 -40 residues longer in the C terminus, forming a "tail" that is absent in CPR. To investigate its function, we removed the 33 and 42 residue C termini from neuronal NOS (nNOS) and endothelial NOS (eNOS), respectively. Both truncated enzymes exhibited cytochrome c reductase activities without calmodulin that were 7-21-fold higher than the nontruncated forms. With calmodulin, the truncated and wild-type enzymes reduced cytochrome c at approximately equal rates. Therefore, calmodulin functioned as a nonessential activator of the wild-type enzymes and a partial noncompetitive inhibitor of the truncated mutants. Truncated nNOS and eNOS plus calmodulin catalyzed NO formation at rates that were 45 and 33%, respectively, those of their intact forms. Without calmodulin, truncated nNOS and eNOS synthesized NO at rates 14 and 20%, respectively, those with calmodulin. By using stopped-flow spectrophotometry, we demonstrated that electron transfer into and between the two flavins is faster in the absence of the C terminus. Although both CPR and intact NOS can exist in a stable, one-electron-reduced semiquinone form, neither of the truncated enzymes do so. We propose negative modulation of FAD-FMN interaction by the C termini of both constitutive NOSs.Nitric-oxide synthases (NOSs) 1 are bidomain, dimeric enzymes that synthesize NO from L-arginine through a series of monooxygenation reactions (for review see Ref. 1). The three isoforms, neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS), produce NO by the same mechanism but play very different physiological roles due to the type of cell where they are located. nNOS, located in neurons in the brain and at neuromuscular junctions, is involved in neurotransmission (2, 3); iNOS, located in macrophages, is involved in the immune response (4, 5); and eNOS, located in endothelial cells, is involved in hemodynamic regulation (6, 7). The NO produced by nNOS and eNOS exerts its effects through the stimulation of guanylate cyclase, whereas the NO produced by iNOS exerts its effects directly or by combining with superoxide anion radical to form peroxynitrite anion, both potent oxidants deleterious to proteins and DNA.The NOSs consist of two domains, a heme and H 4 B-containing oxygenase (or heme) domain, which binds the substrate L-arginine, and a flavin-containing reductase (or flavoprotein) domain, which binds the prosthetic group flavins FAD and FMN and the cofactor NADPH. Electrons are transferred into NOS at the FAD moiety and are subsequently passed through the FMN to the heme domain. A calmodulin-binding region bisects the two domains. Calmodulin (CaM) is required for NO production and mediates the transfer of electrons from the FMN of NOS to the heme domain (8). CaM binds NOS in a 1:1 stoichiometry with very high affinity. CaM binds to sequences within the nNOS, eNOS, and iNOS CaM-binding...

A P8wdomonaa sp., isolated from soil, grew with aniline as its sole carbon source. Washed cells oxidized aniline by an induced enzyme system and liberated ammonia. Washed, aniline-grown organisms oxidized without lag aniline, catechol, p-aminophenol and 0 -and m-toluidine.[4571 TABAE, H. H., CHAMBERS, C. W. & KABLER, P. W. (1964). Microbial metabolism of aromatic compounds. I. Decomposition of phenolic compounds and aromatic hydrocarbons by phenoladapted bacteria. J . Bact. 87, 910. fate of substituted urea herbicides in agricultural soils. Agrorh. J. 47, 93. metabolism of carbohydrates by various Gram-negative bacteria. J . Bad. 66, 24. sources. Arch. Bioclwn. 26 401. compounds. J . Bact. 41, 373.

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