Neuronal nitric-oxide synthase mutant (Ser-1412 --> Asp) demonstrates surprising connections between heme reduction, NO complex formation, and catalysis

Rat neuronal NO synthase (nNOS) contains an Akt-dependent phosphorylation motif in its reductase domain. We mutated a target residue in that site (Ser-1412 to Asp) to mimic phosphorylation and then characterized the mutant using conventional and stopped-flow spectroscopies. Compared with wild-type, S1412D nNOS catalyzed faster cytochrome c and ferricyanide reduction but displayed slower steady-state NO synthesis with greater uncoupling of NADPH oxidation. Paradoxically, the mutant had faster heme reduction, faster heme-NO complex formation, and greater heme-NO complex accumulation at steady state. To understand how these behaviors related to flavin and heme reduction rates, we utilized three soybean calmodulins (CaMs) that supported a range of slower flavin and heme reduction rates in mutant and wild-type nNOS. Reductase activity and two catalytic parameters (speed and amount of heme-NO complex formation) related directly to the speed of flavin and heme reduction. In contrast, steady-state NO synthesis increased, reached a plateau, and then fell at the highest rate of heme reduction that was obtained with S1412D nNOS + CaM. Substituting with soybean CaM slowed heme reduction and increased steady-state NO synthesis by the mutant. We conclude the following. 1) The S1412D mutation speeds electron transfer out of the reductase domain. 2) Faster heme reduction speeds intrinsic NO synthesis but diminishes NO release in the steady state. 3) Heme reduction displays an optimum regarding NO release during steady state. The unique behavior of S1412D nNOS reveals the importance of heme reduction rate in controlling steady-state activity and suggests that nNOS already has a near-optimal rate of heme reduction.     

A proximal tryptophan in NO synthase controls activity by a novel mechanism

The heme of neuronal nitric oxide synthase (nNOS) participates in O2 activation but also binds self-generated NO, resulting in reversible feedback inhibition. We utilized mutagenesis to investigate if a conserved tryptophan residue (Trp409), which engages in pi-stacking with the heme and hydrogen bonds to its axial cysteine ligand, helps control catalysis and regulation by NO. Mutants W409F and W409Y were hyperactive regarding NO synthesis without affecting cytochrome c reduction, reductase-independent N-hydroxyarginine oxidation, or Arg and tetrahydrobiopterin binding. In the absence of Arg electron flux through the heme was slower in the W409 mutants than in wild-type. However, less NO complex accumulated during NO synthesis by the mutants. To understand the mechanism, we compared the kinetics of heme-NO complex formation, rate of heme reduction, kcat prior to and after NO complex formation, NO binding affinity, NO complex stability, and its reaction with O2. During the initial phase of NO synthesis, heme-NO complex formation was three and five times slower in W409F and W409Y, which corresponded to a slower heme reduction. NO complex formation inhibited wild-type turnover 7-fold but reduced mutant turnover less than 2-fold, giving mutants higher steady-state activities. NO binding kinetics were similar among mutants and wild type, although mutants also formed a 417 nm ferrous-NO complex. Oxidation of ferrous-NO complex was seven times faster in mutants than in wild type. We conclude that mutant hyperactivity primarily derives from slower heme reduction and faster oxidation of the heme-NO complex by O2. In this way Trp409 mutations minimize NO feedback inhibition by limiting buildup of the ferrous-NO complex during the steady state. Conservation of W409 among NOS suggests that this proximal Trp may regulate NO feedback inhibition and is important for enzyme physiologic function.     

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

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.     

Variation in anthropometric characters and their relationship with climatic factors: a study on the peoples of Arunachal Pradesh, India

In the present study an attempt has been made to examine the anthropometric variation and the relationship of anthropometric variables with climatic factors among the peoples of Arunachal Pradesh, India. The distance analysis does not seem to provide any interesting corroboration between anthropometric data and the intra- and intergroup relationship. A similar trend has also been noticed while analysing the correlation coefficient between two indices (i.e. the cephalic and total facial index). The present study suggests that the climatic factors are playing an important role in regulating the variation of anthropometric characters among the different population groups of Arunachal Pradesh.     

Village-scale evaluation of mosquito nets treated with a tablet formulation of deltamethrin against malaria vectors

A field trial was carried out in the Sundargarh district of Orissa, India on the efficacy of mosquito nets treated with a tablet formulation of deltamethrin (K-O TAB) against malaria vectors. Treated nets were used in one village, and in the two control villages, one used untreated nets and the other used indoor spraying with DDT, without nets. In this area the primary malaria vectors are Anopheles culicifacies Giles sensu lato (Diptera: Culicidae) and An. fluviatilis James s.l., which are both endophagic and endophilic, and fully susceptible to deltamethrin. Treatment of a 10-m(2) mosquito net with one of the tablets gave a deltamethrin deposit of 25 mg/m(2). Bioassays repeated on domestically used nets over 7 months showed persistence of almost 100% mortality of An. fluviatilis, whereas An. culicifacies showed a decline from 100% to 71% mortality over this period, after which the nets were re-treated and bioassays were not continued. The sum of collections of mosquitoes resting in village houses and those in exit traps and dead on floor sheets showed a reduction in the numbers of the two vector species due to the treated nets, compared with untreated or no nets, but no reduction in other anophelines or Culex species. Large proportions of the collections of the vector and non-vector anophelines were dead on the floor sheets, but among Culex, mortality was delayed. Treated and untreated nets reduced the proportion of anophelines that had blood-fed; the treated nets did so more effectively than the untreated in the case of An. culicifacies and of Culex mosquitoes. In rooms with treated nets a larger proportion of the total collections [dead + live] were in the exit traps, which can be attributed to the excito-repellent effect of deltamethrin. It is easier to pack and handle tablets of insecticide than liquid concentrate and the use of one tablet per net may be preferable to making up a large volume of diluted insecticide and dipping many nets at a time.     

Susceptibility of species A, B, and C of Anopheles culicifacies complex to Plasmodium yoelii yoelii and Plasmodium vinckei petteri infections

The comparative susceptibilities of colonized species A, B, and C of Anopheles culicifacies complex and Anopheles stephensi were determined for 2 rodent malaria parasites Plasmodium vinckei petteri and Plasmodium yoelii yoelii. All the 3 members of the complex were found to support complete sporogony with varying success. Controls, A. stephensi, become readily infected, with >70% developing oocysts. Of the test groups, species A had the highest percentage of mosquitoes with oocysts (>25%) and sporozoites (>15%). Anopheles culicifacies species B were least susceptible; less than 10% had oocysts and sporozoites in the salivary glands. The results demonstrate that A. culicifacies species A is most susceptible and species B is least susceptible to infections with both the parasites.     

Structure of a nitric oxide synthase heme protein from Bacillus subtilis

Eukaryotic nitric oxide synthases (NOSs) produce nitric oxide to mediate intercellular signaling and protect against pathogens. Recently, proteins homologous to mammalian NOS oxygenase domains have been found in prokaryotes and one from Bacillus subtilis (bsNOS) has been demonstrated to produce nitric oxide [Adak, S., Aulak, K. S., and Stuehr, D. J. (2002) J. Biol. Chem. 277, 16167-16171]. We present structures of bsNOS complexed with the active cofactor tetrahydrofolate and the substrate L-arginine (L-Arg) or the intermediate N(omega)-hydroxy-L-arginine (NHA) to 1.9 or 2.2 A resolution, respectively. The bsNOS structure is similar to those of the mammalian NOS oxygenase domains (mNOS(ox)) except for the absence of an N-terminal beta-hairpin hook and zinc-binding region that interact with pterin and stabilize the mNOS(ox) dimer. Changes in patterns of residue conservation between bacterial and mammalian NOSs correlate to different binding modes for pterin side chains. Residue conservation on a surface patch surrounding an exposed heme edge indicates a likely interaction site for reductase proteins in all NOSs. The heme pockets of bsNOS and mNOS(ox) recognize L-Arg and NHA similarly, although a change from Val to Ile beside the substrate guanidinium may explain the 10-20-fold slower dissociation of product NO from the bacterial enzyme. Overall, these structures suggest that bsNOS functions naturally to produce nitrogen oxides from L-Arg and NHA in a pterin-dependent manner, but that the regulation and purpose of NO production by NOS may be quite different in B. subtilis than in mammals.     

Direct evidence for nitric oxide production by a nitric-oxide synthase-like protein from Bacillus subtilis

Nitric-oxide synthases (NOSs) are widely distributed among prokaryotes and eukaryotes and have diverse functions in physiology. Recent genome sequencing revealed NOS-like protein in bacteria, but whether these proteins generate nitric oxide is unknown. We therefore cloned, expressed, and purified a NOS-like protein from Bacillus subtilis (bsNOS) and characterized its catalytic parameters in both multiple and single turnover reactions. bsNOS was dimeric, bound l-Arg and 6R-tetrahydrobiopterin with similar affinity as mammalian NOS, and generated nitrite from l-Arg when incubated with NADPH and a mammalian NOS reductase domain. Stopped-flow analysis showed that ferrous bsNOS reacted with O(2) to form a transient heme Fe(II)O(2) species in the presence of either Arg or the reaction intermediate N-hydroxy-l-arginine. In the latter case, disappearance of the Fe(II)O(2) species was kinetically and quantitatively coupled to formation of a transient heme Fe(III)NO product, which then dissociated to form ferric bsNOS. This behavior mirrors mammalian NOS enzymes and unambiguously shows that bsNOS can generate NO. NO formation required a bound tetrahydropteridine, and the kinetic effects of this cofactor were consistent with it donating an electron to the Fe(II)O(2) intermediate during the reaction. Dissociation of the heme Fe(III)NO product was much slower in bsNOS than in mammalian NOS. This constrains allowable rates of ferric heme reduction by a protein redox partner and underscores the utility of using a tetrahydropteridine electron donor in bsNOS.     

Bio indices for 2,4-D sensitivity between two plant species: <Emphasis Type="Italic">Azolla pinnata</Emphasis> R.Br. and <Emphasis Type="Italic">Vernonia cinerea</Emphasis> L. with their cellular responses

In the present experiment a pteridophytic species Azolla and an angiospermic species Vernonia were evaluated on the basis of cellular reactivity for herbicidal action through ongoing concentrations. Initially, both the species recorded a significant activity of IAA-oxidase as mark of IAA metabolism with herbicidal sensitivity. Still, Vernonia species were more affected on 2,4-D mediated auxin catabolism. The loss of auxin concentrations on the tissues by 2,4-D reaction was also reflected on growth parameters including relative growth rate and chlorophyll biosynthesis. In a dose dependent manner Vernonia plants were more affected with loss of chlorophyll content and decline in relative growth rate. On the other hand, both those parameters were adjusted significantly with 2,4-D accumulation in Azolla. The stability of cellular metabolism was documented by significant down regulation of protein and lipid peroxidation with concomitant moderation to superoxide and hydrogen peroxide accumulation. The later two were more vulnerable to damage in the Vernonia plant with profuse accumulation of protein and lipid peroxidation products. Similarly, tissue specific reaction to superoxide and hydrogen peroxide accumulation were distinctly demarcated in two species significantly. As a whole, the cellular responses and metabolite distribution to 2,4-D sensitization are the features to describe bio-indices for aquatic fern species Azolla with comparison to angiospermic species Vernonia.     

Synthesis and antimicrobial activity of new 1,2,4-triazole and 1,3,4-thiadiazole derivatives

In this study, new 3-[(1(2H)-phthalazinone-2-yl(methyl/ethyl]-4-aryl-1,2,4-triazole-5-thione and 2-[[1(2H)-phthalazinone-2-yl]methyl/ethyl]-5-arylamino-1,3,4-thiadiazole derivatives were synthesized. Antimicrobial properties of the title compounds were investigated against two Gram (+) bacteria (S. aureus, B. subtilis), two Gram ( - ) bacteria (P. aeruginosa, E. coli) and two yeast-like fungi (C. albicans and C. parapsilosis) using the broth microdilution method. Generally the compounds were found to be active against B. subtilis and the fungi. Derivatives carrying a 1,3,4-thiadiazole ring generally showed higher antimicrobial activity against B. subtilis and the fungi when compared to other synthesized compounds.