Search

Background: Trypanothione synthetase catalyzes the conjugation of spermidine with two GSH molecules to form trypanothione. Results: The kinetic parameters were measured under in vivo-like conditions. A mathematical model was developed describing the entire kinetic profile. Conclusion: Trypanothione synthetase is affected by substrate and product inhibition. Significance: The combined kinetic and modeling approaches provided a so far unprecedented insight in the mechanism of this parasite-specific enzyme., In pathogenic trypanosomes, trypanothione synthetase (TryS) catalyzes the synthesis of both glutathionylspermidine (Gsp) and trypanothione (bis(glutathionyl)spermidine (T(SH)2)). Here we present a thorough kinetic analysis of Trypanosoma brucei TryS in a newly developed phosphate buffer system at pH 7.0 and 37 °C, mimicking the physiological environment of the enzyme in the cytosol of bloodstream parasites. Under these conditions, TryS displays Km values for GSH, ATP, spermidine, and Gsp of 34, 18, 687, and 32 μm, respectively, as well as Ki values for GSH and T(SH)2 of 1 mm and 360 μm, respectively. As Gsp hydrolysis has a Km value of 5.6 mm, the in vivo amidase activity is probably negligible. To obtain deeper insight in the molecular mechanism of TryS, we have formulated alternative kinetic models, with elementary reaction steps represented by linear kinetic equations. The model parameters were fitted to the extensive matrix of steady-state data obtained for different substrate/product combinations under the in vivo-like conditions. The best model describes the full kinetic profile and is able to predict time course data that were not used for fitting. This system's biology approach to enzyme kinetics led us to conclude that (i) TryS follows a ter-reactant mechanism, (ii) the intermediate Gsp dissociates from the enzyme between the two catalytic steps, and (iii) T(SH)2 inhibits the enzyme by remaining bound at its product site and, as does the inhibitory GSH, by binding to the activated enzyme complex. The newly detected concerted substrate and product inhibition suggests that TryS activity is tightly regulated.

The aim of this study was to explore the role of vitamin D in rheumatoid arthritis (RA) pathogenesis by investigating the enrichment of vitamin D response elements (VDREs) in confirmed RA susceptibility loci and testing variants associated with vitamin D levels for association with RA. Bioinformatically, VDRE genomic positions were overlaid with non-HLA (human leukocyte antigen)-confirmed RA susceptibility regions. The number of VDREs at RA loci was compared to a randomly selected set of genomic loci to calculate an average relative risk (RR). Single-nucleotide polymorphisms (SNPs) in the DHCR7/NADSYN1 (nicotinamide adenine dinucleotide synthase 1) and CYP2R1 loci, previously associated with circulating vitamin D levels, were tested in UK RA cases (n=3870) and controls (n=8430). Significant enrichment of VDREs was seen at RA loci (P=9.23 × 10(-8)) when regions were defined either by gene (RR 5.50) or position (RR 5.86). SNPs in the DHCR7/NADSYN1 locus showed evidence of positive association with RA, rs4944076 (P=0.008, odds ratio (OR) 1.14, 95% confidence interval (CI) 1.03-1.24). The significant enrichment of VDREs at RA-associated loci and the modest association of variants in loci-controlling levels of circulating vitamin D supports the hypothesis that vitamin D has a role in the development of RA.

In mice cynaropicrin (CYN) potently inhibits the proliferation of Trypanosoma brucei-the causative agent of Human African Trypanosomiasis-by a so far unknown mechanism. We hypothesized that CYNs α,β-unsaturated methylene moieties act as Michael acceptors for glutathione (GSH) and trypanothione (T(SH)2), the main low molecular mass thiols essential for unique redox metabolism of these parasites. The analysis of this putative mechanism and the effects of CYN on enzymes of the T(SH)2 redox metabolism including trypanothione reductase, trypanothione synthetase, glutathione-S-transferase, and ornithine decarboxylase are shown. A two step extraction protocol with subsequent UPLC-MS/MS analysis was established to quantify intra-cellular CYN, T(SH)2, GSH, as well as GS-CYN and T(S-CYN)2 adducts in intact T. b. rhodesiense cells. Within minutes of exposure to CYN, the cellular GSH and T(SH)2 pools were entirely depleted, and the parasites entered an apoptotic stage and died. CYN also showed inhibition of the ornithine decarboxylase similar to the positive control eflornithine. Significant interactions with the other enzymes involved in the T(SH)2 redox metabolism were not observed. Alongside many other biological activities sesquiterpene lactones including CYN have shown antitrypanosomal effects, which have been postulated to be linked to formation of Michael adducts with cellular nucleophiles. Here the interaction of CYN with biological thiols in a cellular system in general, and with trypanosomal T(SH)2 redox metabolism in particular, thus offering a molecular explanation for the antitrypanosomal activity is demonstrated. At the same time, the study provides a novel extraction and analysis protocol for components of the trypanosomal thiol metabolism.

International audience; Trypanothione synthetase is an essential enzyme for kinetoplastid parasites which cause highly disabling and fatal diseases in humans and animals. Inspired by the observation that N(5)-substituted paullones inhibit the trypanothione synthetase from the related parasite Leishmania infantum, we designed and synthesized a series of new derivatives. Although none of the new compounds displayed strong inhibition of Trypanosoma brucei trypanothione synthetase, several of them caused a remarkable growth inhibition of cultivated Trypanosoma brucei bloodstream forms. The most potent congener 3a showed antitrypanosomal activity in double digit nanomolar concentrations and a selectivity index of three orders of magnitude versus murine macrophage cells.

Herein we report a practical synthetic route to the lasso peptide lassomycin () and C-terminal variant lassomycin-amide (). The biological evaluation of peptides and against Mycobacterium tuberculosis revealed that neither had any activity against this bacterium. This lack of biological activity has led us to propose that naturally occurring lassomycin may actually exhibit a standard lasso peptide threaded conformation rather than the previously reported unthreaded structure.

Background The search for novel chemical entities targeting essential and parasite-specific pathways is considered a priority for neglected diseases such as trypanosomiasis and leishmaniasis. The thiol-dependent redox metabolism of trypanosomatids relies on bis-glutathionylspermidine [trypanothione, T(SH)2], a low molecular mass cosubstrate absent in the host. In pathogenic trypanosomatids, a single enzyme, trypanothione synthetase (TryS), catalyzes trypanothione biosynthesis, which is indispensable for parasite survival. Thus, TryS qualifies as an attractive drug target candidate. Methodology/Principal Finding A library composed of 144 compounds from 7 different families and several singletons was screened against TryS from three major pathogen species (Trypanosoma brucei, Trypanosoma cruzi and Leishmania infantum). The screening conditions were adjusted to the TryS´ kinetic parameters and intracellular concentration of substrates corresponding to each trypanosomatid species, and/or to avoid assay interference. The screening assay yielded suitable Z’ and signal to noise values (≥0.85 and ~3.5, respectively), and high intra-assay reproducibility. Several novel chemical scaffolds were identified as low μM and selective tri-tryp TryS inhibitors. Compounds displaying multi-TryS inhibition (N,N'-bis(3,4-substituted-benzyl) diamine derivatives) and an N5-substituted paullone (MOL2008) halted the proliferation of infective Trypanosoma brucei (EC50 in the nM range) and Leishmania infantum promastigotes (EC50 = 12 μM), respectively. A bis-benzyl diamine derivative and MOL2008 depleted intracellular trypanothione in treated parasites, which confirmed the on-target activity of these compounds. Conclusions/Significance Novel molecular scaffolds with on-target mode of action were identified as hit candidates for TryS inhibition. Due to the remarkable species-specificity exhibited by tri-tryp TryS towards the compounds, future optimization and screening campaigns should aim at designing and detecting, respectively, more potent and broad-range TryS inhibitors., Author Summary Parasites from the genus Trypanosoma and Leishmania are etiologic agents for a group of neglected diseases with high morbidity and mortality rates in the developing world. Inasmuch as vaccine development is hampered by the successful mechanisms employed by the pathogens to evade the host immune response, chemotherapy remains as a safe option to fight these diseases. However, new drugs with better pharmacological performance (i.e. safety, efficacy and ease of administration) than those in current use are urgently needed. The thiol-redox metabolism of trypanosomatids offers an excellent opportunity for the development of more selective and efficacious medicines because it depends on a molecule, trypanothione (a bis-glutathionyl derivative of spermidine), unique and indispensable to the pathogens. Here we report the identification of novel inhibitors of trypanothione synthetase from three major trypanosomatid species of medical and veterinary relevance. Although highly conserved in sequence, trypanothione synthetases display significant species-specifity towards compounds, pointing to structural differences as determinants of ligand selectivity. Most of the active compounds presented two-digit μM inhibitory activity and serve as primary scaffolds to develop more potent inhibitors. Among them, N,N'-bis(benzyl)-substituted diamine and paullone derivatives are interesting candidates because of their potent and/or selective anti-trypanosomal and anti-trypanothione synthetase activity.

The essential coenzyme NAD plays important roles in metabolic reactions and cell regulation in all organisms. As such, NAD synthesis has been investigated as a source for novel antibacterial targets. Cross-species genomics-based reconstructions of NAD metabolism in group A streptococci (GAS), combined with focused experimental testing in Streptococcus pyogenes, led to a better understanding of NAD metabolism in the pathogen. The predicted niacin auxotrophy was experimentally verified, as well as the essential role of the nicotinamidase PncA in the utilization of nicotinamide (Nm). PncA is dispensable in the presence of nicotinate (Na), ruling it out as a viable antibacterial target. The function of the "orphan" NadC enzyme, which is uniquely present in all GAS species despite the absence of other genes of NAD de novo synthesis, was elucidated. Indeed, the quinolinate (Qa) phosphoribosyltransferase activity of NadC from S. pyogenes allows the organism to sustain growth when Qa is present as a sole pyridine precursor. Finally, the redundancy of functional upstream salvage pathways in GAS species narrows the choice of potential drug targets to the two indispensable downstream enzymes of NAD synthesis, nicotinate adenylyltransferase (NadD family) and NAD synthetase (NadE family). Biochemical characterization of NadD confirmed its functional role in S. pyogenes, and its potential as an antibacterial target was supported by inhibition studies with previously identified class I inhibitors of the NadD enzyme family. One of these inhibitors efficiently inhibited S. pyogenes NadD (sp.NadD) in vitro (50% inhibitory concentration [IC(50)], 15 μM), exhibiting a noncompetitive mechanism with a K(i) of 8 μM.

Glutathionylspermidine synthetase/amidase (Gss) and the encoding gene (gss) have only been studied in Escherichia coli and several members of the Kinetoplastida phyla. In the present article, we have studied the phylogenetic distribution of Gss and have found that Gss sequences are largely limited to certain bacteria and Kinetoplastids and are absent in a variety of invertebrate and vertebrate species, Archea, plants, and some Eubacteria. It is striking that almost all of the 75 Enterobacteria species that have been sequenced contain sequences with very high degree of homology to the E. coli Gss protein. To find out the physiological significance of glutathionylspermidine in E. coli, we have performed global transcriptome analyses. The microarray studies comparing gss(+) and Δgss strains of E. coli show that a large number of genes are either up-regulated (76 genes more than threefold) or down-regulated (35 genes more than threefold) by the loss of the gss gene. Most significant categories of up-regulated genes include sulfur utilization, glutamine and succinate metabolism, polyamine and arginine metabolism, and purine and pyrimidine metabolism.

Biosynthesis and the use of trypanothione, a redox metabolite of parasitic trypanosomatids, are reviewed here with special emphasis on the development of trypanocidal drugs. This metabolic system is unique to and essential for the protozoal parasites. Selective inhibition of key elements of trypanothione metabolism, therefore, promises eradication of the parasites without affecting the host. Considering the metabolic importance and drugability of system components, inhibition of the enzymes for regeneration and de novo synthesis of trypanothione is rated as the most promising approach, while related peroxidases and redoxins are disregarded as targets because of limited chances to achieve selective inhibition. The organizational need to exploit the accumulating knowledge of trypanosomatid metabolism for medical practice is briefly addressed.

International audience; Suranadi IW, Demaison L, Chate V, Peltier S, Richardson M, Leverve X. An increase in the redox state during reperfusion contributes to the cardioprotective effect of GIK solution. J Appl Physiol 113: 775-784, 2012. First published July 12, 2012; doi: 10.1152/japplphysiol.01153.2011.-This study aimed at determining whether glucose-insulin-potassium (GIK) solutions modify the NADH/NAD(+) ratio during postischemic reperfusion and whether their cardioprotective effect can be attributed to this change in part through reduction of the mitochondrial reactive oxygen species (ROS) production. The hearts of 72 rats were perfused with a buffer containing glucose (5.5 mM) and hexanoate (0.5 mM). They were maintained in normoxia for 30 min and then subjected to low-flow ischemia (0.5% of the preischemic coronary flow for 20 min) followed by reperfusion (45 min). From the beginning of ischemia, the perfusate was subjected to various changes: enrichment with GIK solution, enrichment with lactate (2 mM), enrichment with pyruvate (2 mM), enrichment with pyruvate (2 mM) plus ethanol (2 mM), or no change for the control group. Left ventricular developed pressure, heart rate, coronary flow, and oxygen consumption were monitored throughout. The lactate/pyruvate ratio of the coronary effluent, known to reflect the cytosolic NADH/NAD(+) ratio and the fructose-6-phosphate/ dihydroxyacetone-phosphate (F6P/DHAP) ratio of the reperfused myocardium, were evaluated. Mitochondrial ROS production was also estimated. The GIK solution improved the recovery of mechanical function during reperfusion. This was associated with an enhanced cytosolic NADH/NAD(+) ratio and reduced mitochondrial ROS production. The cardioprotection was also observed when the hearts were perfused with fluids known to increase the cytosolic NADH/NAD(+) ratio (lactate, pyruvate plus ethanol) compared with the other fluids (control and pyruvate groups). The hearts with a high mechanical recovery also displayed a low F6P/DHAP ratio, suggesting that an accelerated glycolysis rate may be responsible for increased cytosolic NADH production. In conclusion, the cardioprotection induced by GIK solutions could occur through an increase in the cytosolic NADH/NAD(+) ratio, leading to a decrease in mitochondrial ROS production.

Theanine synthetase (TS) is an enzyme involved in theanine biosynthesis in tea plants. Recent studies have revealed that theanine biosynthesis, derived from nitrogen metabolism in tea (Camellia sinensis L.) plants, could be influenced by salt treatment. We have characterized CsTS at the molecular and biochemical level. The expression pattern of CsTS protein was examined by western blot using a self-prepared polyclonal antibody with high specificity and sensitivity. The effect of salt treatment on the levels of theanine synthesis was investigated in this study. Levels of theanine and the total free amino acids were gradually increased in shoots, and reached the maximum on the 8th day after treatment (DAT). The immunoblotting analysis suggested the accumulation of CsTS protein had increased gently up to 8 DAT, and subsequently declined, both in roots and shoots, which is one of the main evidences that resulted in the variation of theanine concentration under salt treatment. Together, these data revealed that theanine synthesis takes place both in root and shoot and CsTS accumulation is positively affected by salt treatment.

The amide synthase of the geldanamycin producer, Streptomyces hygroscopicus, shows a broader chemoselectivity than the corresponding amide synthase present in Actinosynnema pretiosum, the producer of the highly cytotoxic ansamycin antibiotics, the ansamitocins. This was demonstrated when blocked mutants of both strains incapable of biosynthesizing 3-amino-5-hydroxybenzoic acid (AHBA), the polyketide synthase starter unit of both natural products, were supplemented with 3-amino-5-hydroxymethylbenzoic acid instead. Unlike the ansamitocin producer A. pretiosum, S. hygroscopicus processed this modified starter unit not only to the expected 19-membered macrolactams but also to ring enlarged 20-membered macrolactones. The former mutaproducts revealed the sequence of transformations catalyzed by the post-PKS tailoring enzymes in geldanamycin biosynthesis. The unprecedented formation of the macrolactones together with molecular modeling studies shed light on the mode of action of the amide synthase responsible for macrocyclization. Obviously, the 3-hydroxymethyl substituent shows similar reactivity and accessibility toward C-1 of the seco-acid as the arylamino group, while phenolic hydroxyl groups lack this propensity to act as nucleophiles in the macrocyclization. The promiscuity of the amide synthase of S. hygroscopicus was further demonstrated by successful feeding of four other m-hydroxymethylbenzoic acids, leading to formation of the expected 20-membered macrocycles. Good to moderate antiproliferative activities were encountered for three of the five new geldanamycin derivatives, which matched well with a competition assay for Hsp90α.

Trypanothione synthetase, a validated drug target, synthesizes trypanothione from glutathione and spermidine. Here we report the gene cloning, expression, characterization and inhibition studies of trypanothione synthetase from Leishmania donovani (LdTryS). The purified recombinant LdTryS enzyme obeyed Michaelis-Menten kinetics. High substrate inhibition was observed with glutathione (Km=33.24 μm, kcat=1.3 s-1, Ki=866 μm). The enzyme shows simple hyperbolic kinetics with fixed glutathione concentration and with other substrates limiting Km values for Mg. ATP and spermidine of 14.2 μm and 139.6 μm, respectively. LdTryS was also screened for inhibitors. Tomatine, conessine, uvaol and betulin were identified as inhibitors of the enzyme and were tested for leishmanicidal activity. Finally, the effect of LdTryS inhibitors on redox homeostasis of the parasite gives a broader picture of their action against leishmaniasis.

Better drugs are urgently needed for the treatment of African sleeping sickness. We tested a series of promising anticancer agents belonging to the 4-substituted 4-hydroxycyclohexa-2,5-dienones class ("quinols") and identified several with potent trypanocidal activity (EC(50) < 100 nM). In mammalian cells, quinols are proposed to inhibit the thioredoxin/thioredoxin reductase system, which is absent from trypanosomes. Studies with the prototypical 4-benzothiazole-substituted quinol, PMX464, established that PMX464 is rapidly cytocidal, similar to the arsenical drug, melarsen oxide. Cell lysis by PMX464 was accelerated by addition of sublethal concentrations of glucose oxidase implicating oxidant defenses in the mechanism of action. Whole cells treated with PMX464 showed a loss of trypanothione (T(SH)(2)), a unique dithiol in trypanosomes, and tryparedoxin peroxidase (TryP), a 2-Cys peroxiredoxin similar to mammalian thioredoxin peroxidase. Enzyme assays revealed that T(SH)(2), TryP, and a glutathione peroxidase-like tryparedoxin-dependent peroxidase were inhibited in time- and concentration-dependent manners. The inhibitory activities of various quinol analogues against these targets showed a good correlation with growth inhibition of Trypanosoma brucei. The monothiols glutathione and L-cysteine bound in a 2:1 ratio with PMX464 with K(d) values of 6 and 27 μM, respectively, whereas T(SH)(2) bound more tightly in a 1:1 ratio with a K(d) value of 430 nM. Overexpression of trypanothione synthetase in T. brucei decreased sensitivity to PMX464 indicating that the key metabolite T(SH)(2) is a target for quinols. Thus, the quinol pharmacophore represents a novel lead structure for the development of a new drug against African sleeping sickness.

The primary circulating form of vitamin D, 25-hydroxy-vitamin D [25(OH)D], is associated with multiple medical outcomes, including rickets, osteoporosis, multiple sclerosis and cancer. In a genome-wide association study (GWAS) of 4501 persons of European ancestry drawn from five cohorts, we identified single-nucleotide polymorphisms (SNPs) in the gene encoding group-specific component (vitamin D binding) protein, GC, on chromosome 4q12-13 that were associated with 25(OH)D concentrations: rs2282679 (P = 2.0 × 10−30), in linkage disequilibrium (LD) with rs7041, a non-synonymous SNP (D432E; P = 4.1 × 10−22) and rs1155563 (P = 3.8 × 10−25). Suggestive signals for association with 25(OH)D were also observed for SNPs in or near three other genes involved in vitamin D synthesis or activation: rs3829251 on chromosome 11q13.4 in NADSYN1 [encoding nicotinamide adenine dinucleotide (NAD) synthetase; P = 8.8 × 10−7], which was in high LD with rs1790349, located in DHCR7, the gene encoding 7-dehydrocholesterol reductase that synthesizes cholesterol from 7-dehydrocholesterol; rs6599638 in the region harboring the open-reading frame 88 (C10orf88) on chromosome 10q26.13 in the vicinity of ACADSB (acyl-Coenzyme A dehydrogenase), involved in cholesterol and vitamin D synthesis (P = 3.3 × 10−7); and rs2060793 on chromosome 11p15.2 in CYP2R1 (cytochrome P450, family 2, subfamily R, polypeptide 1, encoding a key C-25 hydroxylase that converts vitamin D3 to an active vitamin D receptor ligand; P = 1.4 × 10−5). We genotyped SNPs in these four regions in 2221 additional samples and confirmed strong genome-wide significant associations with 25(OH)D through meta-analysis with the GWAS data for GC (P = 1.8 × 10−49), NADSYN1/DHCR7 (P = 3.4 × 10−9) and CYP2R1 (P = 2.9 × 10−17), but not C10orf88 (P = 2.4 × 10−5).

NAD(+) synthetase catalyzes the formation of NAD(+) from ATP, nicotinic acid adenine dinucleotide and ammonia. Glutamine-dependent NAD(+) synthetase obtains ammonia through the hydrolysis of glutamine to glutamate, which takes place in the glutaminase domain. The ammonia is subsequently transported to the synthetase domain through an interdomain ammonia tunnel. NAD(+) synthetase from the thermophilic bacteria Thermotoga maritima was cloned and expressed. Steady-state kinetics and stoichiometric analysis of product formation revealed an enzyme that is significantly inefficient in the synchronization of the two active sites resulting in wasteful hydrolysis of glutamine and that is not specific for glutamine over ammonia. Phylogenetic analysis of glutamine-dependent NAD(+) synthetases identifies three main groups remotely related. The T. maritima NAD(+) synthetase's group is proposed to represent the ancestral group based on the phylogenetic analysis and on the kinetic characterizations. The phylogenetic results nicely correlate also with the degree of catalytic efficiency measured for M. tuberculosis, S. cerevisiae and T. maritima NAD(+) synthetases. Furthermore, the data here reported in combination with structural data available for glutamine-dependent NAD(+) synthetase lays the foundation for further investigation on the mechanism of active site coupling in these enzymes.

Virtual screening was employed to identify new drug-like inhibitors of NAD synthetase (NADs) as antibacterial agents. Four databases of commercially available compounds were docked against three subsites of the NADs active site using FlexX in conjunction with CScore. Over 200 commercial compounds were purchased and evaluated in enzyme inhibition and antibacterial assays. 18 compounds inhibited NADs at or below 100 microM (7.6% hit rate), and two were selected for future SAR studies.

NAD(P) biosynthetic pathways can be considered a generous source of enzymatic targets for drug development. Key reactions for NAD(P) biosynthesis in all organisms, common to both de novo and salvage routes, are catalyzed by NMN/NaMN adenylyltransferase (NMNAT), NAD synthetase (NADS), and NAD kinase (NADK). These reactions represent a three-step pathway, present in the vast majority of living organisms, which is responsible for the generation of both NAD and NADP cellular pools. The validation of these enzymes as drug targets is based on their essentiality and conservation among a large variety of pathogenic microorganisms, as well as on their differential structural features or their differential metabolic contribution to NAD(P) homeostasis between microbial and human cell types. This review describes the structural and functional properties of eubacterial and human enzymes endowed with NMNAT, NADS, and NADK activities, as well as with nicotinamide phosphoribosyltransferase (NamPRT) and nicotinamide riboside kinase (NRK) activities, highlighting the species-related differences, with emphasis on their relevance for drug design. In addition, since the overall NMNAT activity in humans is accounted by multiple isozymes differentially involved in the metabolic activation of antineoplastic compounds, their individual diagnostic value for early therapy optimization is outlined. The involvement of human NMNAT in neurodegenerative disorders and its role in neuroprotection is also discussed.

NAD(+) is an essential metabolite both as a cofactor in energy metabolism and redox homeostasis and as a regulator of cellular processes. In contrast to humans, Mycobacterium tuberculosis NAD(+) biosynthesis is absolutely dependent on the activity of a multifunctional glutamine-dependent NAD(+) synthetase, which catalyzes the ATP-dependent formation of NAD(+) at the synthetase domain using ammonia derived from L-glutamine in the glutaminase domain. Here we report the kinetics and structural characterization of M. tuberculosis NAD(+) synthetase. The kinetics data strongly suggest tightly coupled regulation of the catalytic activities. The structure, the first of a glutamine-dependent NAD(+) synthetase, reveals a homooctameric subunit organization suggesting a tight dependence of catalysis on the quaternary structure, a 40-A intersubunit ammonia tunnel and structural elements that may be involved in the transfer of information between catalytic sites.

The bifunctional trypanothione synthetase-amidase catalyzes biosynthesis and hydrolysis of the glutathione-spermidine adduct trypanothione, the principal intracellular thiol-redox metabolite in parasitic trypanosomatids. These parasites are unique with regard to their reliance on trypanothione to determine intracellular thiol-redox balance in defense against oxidative and chemical stress and to regulate polyamine levels. Enzymes involved in trypanothione biosynthesis provide essential biological activities, and those absent from humans or for which orthologues are sufficiently distinct are attractive targets to underpin anti-parasitic drug discovery. The structure of Leishmania major trypanothione synthetase-amidase, determined in three crystal forms, reveals two catalytic domains. The N-terminal domain, a cysteine, histidine-dependent amidohydrolase/peptidase amidase, is a papain-like cysteine protease, and the C-terminal synthetase domain displays an ATP-grasp family fold common to C:N ligases. Modeling of substrates into each active site provides insight into the specificity and reactivity of this unusual enzyme, which is able to catalyze four reactions. The domain orientation is distinct from that observed in a related bacterial glutathionylspermidine synthetase. In trypanothione synthetase-amidase, the interactions formed by the C terminus, binding in and restricting access to the amidase active site, suggest that the balance of ligation and hydrolytic activity is directly influenced by the alignment of the domains with respect to each other and implicate conformational changes with amidase activity. The potential inhibitory role of the C terminus provides a mechanism to control relative levels of the critical metabolites, trypanothione, glutathionylspermidine, and spermidine in Leishmania.

Glutathionylspermidine is an intermediate formed in the biosynthesis of trypanothione, an essential metabolite in defence against chemical and oxidative stress in the Kinetoplastida. The kinetic mechanism for glutathionylspermidine synthetase (EC 6.3.1.8) from Crithidia fasciculata (CfGspS) obeys a rapid equilibrium random ter-ter model with kinetic constants K(GSH) = 609 microM, K(Spd) = 157 microM and K(ATP) = 215 microM. Phosphonate and phosphinate analogues of glutathionylspermidine, previously shown to be potent inhibitors of GspS from Escherichia coli, are equally potent against CfGspS. The tetrahedral phosphonate acts as a simple ground state analogue of glutathione (GSH) (K(i) approximately 156 microM), whereas the phosphinate behaves as a stable mimic of the postulated unstable tetrahedral intermediate. Kinetic studies showed that the phosphinate behaves as a slow-binding bisubstrate inhibitor [competitive with respect to GSH and spermidine (Spd)] with rate constants k(3) (on rate) = 6.98 x 10(4) M(-1) x s(-1) and k(4) (off rate) = 1.3 x 10(-3) s(-1), providing a dissociation constant K(i) = 18.6 nM. The phosphinate analogue also inhibited recombinant trypanothione synthetase (EC 6.3.1.9) from C. fasciculata, Leishmania major, Trypanosoma cruzi and Trypanosoma brucei with K(i)(app) values 20-40-fold greater than that of CfGspS. This phosphinate analogue remains the most potent enzyme inhibitor identified to date, and represents a good starting point for drug discovery for trypanosomiasis and leishmaniasis.

Nicotinic acid adenine dinucleotide phosphate (NAADP) has been shown to mobilize Ca(2+) from intracellular stores in a wide variety of organisms, ranging from plants to humans. We have developed a novel enzyme cycling assay for NAADP that involves coupled reactions catalyzed by four enzymes. In this system, NAADP is first converted into nicotinic acid adenine dinucleotide (NAAD) by alkaline phosphatase, after which the NAAD is converted to NAD, AMP, and PPi by NAD synthetase (NADS) in the presence of ATP and ammonia. The NAD is then amplified using an enzyme cycling system driven by glucose dehydrogenase and diaphorase. The resultant formation of formazan dye is measured spectrophotometrically based on the increase in absorbance at 450 nm. Using this method, NAADP (20-400 nM) was assayed, and a highly linear correlation was obtained between the NAADP concentration and the increase in absorbance at 450 nm. The cycling rate was approximately 95 cycles/min. In addition, the within-run coefficients of variation (CVs) for 25, 50, and 100 nM NAADP solutions were 9.33, 4.86, and 3.13%, respectively. Interference by NAD analogs (e.g., NAAD, NADP) in the sample was eliminated prior to running the assay by treating the sample with NADS and NAD nucleosidase (NADase). In sum, our findings indicate this enzyme cycling assay to be readily applicable for determination for NAADP in a variety of biological samples and to be particularly appropriate for use with an autoanalyzer.

In a previous screen for Mycobacterium tuberculosis mutants that are hypersusceptible to reactive nitrogen intermediates (RNI), two genes associated with the M. tuberculosis proteasome were identified. One of these genes, pafA ( p roteasome a ccessory f actor A), encodes a protein of unknown function. In this work, we determined that pafA is in an operon with two additional genes, pafB and pafC . In order to assess the contribution of these genes to RNI resistance, we isolated mutants with transposon insertions in pafB and pafC . In contrast to the pafA mutant, the pafB and pafC mutants were not severely sensitized to RNI, but pafB and pafC were nonetheless required for full RNI resistance. We also found that PafB and PafC interact with each other and that each is likely required for the stability of the other protein in M. tuberculosis . Finally, we show that the presence of PafA, but not PafB or PafC, regulates the steady-state levels of three proteasome substrates. Taken together, these data demonstrate that PafA, but not PafB or PafC, is critical for maintaining the steady-state levels of known proteasome substrates, whereas all three proteins appear to play a role in RNI resistance.

NAD is a well-known co-enzyme that mediates hundreds of redox reactions and is the basis of various processes regulating cell responses to different environmental and developmental cues. The regulatory mechanism that determines the amount of cellular NAD and the rate of NAD metabolism remains unclear. We created Arabidopsis thaliana plants overexpressing the NAD synthase (NADS) gene that participates in the final step of NAD biosynthesis. NADS overexpression enhanced the activity of NAD biosynthesis but not the amounts of NAD+, NADH, NADP+ or NADPH. However, the amounts of some intermediates were elevated, suggesting that NAD metabolism increased. The NAD redox state was greatly facilitated by an imbalance between NAD generation and degradation in response to bolting. Metabolite profiling and transcriptional analysis revealed that the drastic modulation of NAD redox homeostasis increased tricarboxylic acid flux, causing the ectopic generation of reactive oxygen species. Vascular bundles suffered from oxidative stress, leading to a malfunction in amino acid and organic acid transportation that caused early wilting of the flower stalk and shortened plant longevity, probably due to malnutrition. We concluded that the mechanism regulating the balance between NAD synthesis and degradation is important in the systemic plant response to developmental cues during the growth-phase transition.

Chagas disease is a parasitic infection caused by the protozoa Trypanosoma cruzi that affects about 6 million people in Latin America. Despite its sanitary importance, there are currently only two drugs available for treatment: benznidazole and nifurtimox, both exhibiting serious adverse effects and limited efficacy in the chronic stage of the disease. Polyamines are ubiquitous to all living organisms where they participate in multiple basic functions such as biosynthesis of nucleic acids and proteins, proliferation and cell differentiation. T. cruzi is auxotroph for polyamines, which are taken up from the extracellular medium by efficient transporters and, to a large extent, incorporated into trypanothione (bis-glutathionylspermidine), the major redox cosubstrate of trypanosomatids. From a 268-compound database containing polyamine analogs with and without inhibitory effect on T. cruzi we have inferred classificatory models that were later applied in a virtual screening campaign to identify anti-trypanosomal compounds among drugs already used for other therapeutic indications (i.e. computer-guided drug repositioning) compiled in the DrugBank and Sweetlead databases. Five of the candidates identified with this strategy were evaluated in cellular models from different pathogenic trypanosomatids (T. cruzi wt, T. cruzi PAT12, T. brucei and Leishmania infantum), and in vitro models of aminoacid/polyamine transport assays and trypanothione synthetase inhibition assay. Triclabendazole, sertaconazole and paroxetine displayed inhibitory effects on the proliferation of T. cruzi (epimastigotes) and the uptake of putrescine by the parasite. They also interfered with the uptake of others aminoacids and the proliferation of infective T. brucei and L. infantum (promastigotes). Trypanothione synthetase was ruled out as molecular target for the anti-parasitic activity of these compounds.

Nicotinamide adenine dinucleotide synthetase enzyme (NadE) catalyzes the amination of nicotinic acid adenine dinucleotide (NaAD) to form NAD(+). This reaction represents the last step in the majority of the NAD(+) biosynthetic routes described to date. NadE enzymes typically use either glutamine or ammonium as amine nitrogen donor, and the reaction is energetically driven by ATP hydrolysis. Given the key role of NAD(+) in bacterial metabolism, NadE has attracted considerable interest as a potential target for the development of novel antibiotics. The plant-associative nitrogen-fixing bacteria Herbaspirillum seropedicae encodes two putative NadE, namely nadE1 and nadE2. The nadE1 gene is linked to glnB encoding the signal transduction protein GlnB. Here we report the purification and in vitro characterization of H. seropedicae NadE1. Gel filtration chromatography analysis suggests that NadE1 is an octamer. The NadE1 activity was assayed in vitro, and the Michaelis-Menten constants for substrates NaAD, ATP, glutamine and ammonium were determined. Enzyme kinetic and in vitro substrate competition assays indicate that H. seropedicae NadE1 uses glutamine as a preferential nitrogen donor.

Trypanosomatids are the causative agents of African sleeping sickness, Chagas' disease, and the different forms of leishmaniasis. This family of protozoan parasite possesses a trypanothione-based redox metabolism that provides the reducing equivalents for various vital processes such as the biosynthesis of DNA precursors and the detoxification of hydroperoxides. Almost all enzymes of the redox pathway proved to be essential and therefore fulfil one crucial prerequisite for a putative drug target. Trypanothione synthetase and trypanothione reductase are present in all trypanosomatids but absent from the mammalian host which, in addition to the essentiality, renders them highly specific. The chemotherapy research on both enzymes is further supported by the availability of high-throughput screening techniques and crystal structures. In this review we focus on the recent advances and limitations in the development of lead compounds targeting trypanothione synthetase and trypanothione reductase. We present an overview of the available inhibitors and discuss future perspectives including other components of the parasite-specific redox pathway.

Nosiheptide is a parent compound of thiopeptide family that exhibit potent activities against various bacterial pathogens. Its C-terminal amide formation is catalyzed by NosA, which is an unusual strategy for maturating certain thiopeptides by processing their precursor peptides featuring a serine extension. We here report the crystal structure of truncated NosA1-111 variant, revealing three key elements, including basic lysine 49 (K49), acidic glutamic acid 101 (E101) and flexible C-terminal loop NosA112-151, are crucial to the catalytic terminal amide formation in nosiheptide biosynthesis. The side-chain of residue K49 and the C-terminal loop fasten the substrate through hydrogen bonds and hydrophobic interactions. The side-chain of residue E101 enhances nucleophilic attack of H2O to the methyl imine intermediate, leading to Cα-N bond cleavage and nosiheptide maturation. The sequence alignment of NosA and its homologs NocA, PbtH, TpdK and BerI and the enzymatic assay suggest that the mechanistic studies on NosA present an intriguing paradigm about how NosA family members function during thiopeptide biosynthesis.

Trypanothione synthetase (TryS) is a protein unique to the Kinetoplastida and has been shown to be essential in Trypanosoma brucei by RNAi techniques [1,2]. TryS is the sole enzyme responsible for the biosynthesis of trypanothione in the human pathogenic parasites T. brucei, Trypanosoma cruzi and Leishmania major [3–5]. The protein is thought to have two domains: an ATP-dependent synthetase domain that generates the intermediate, glutathionylspermidine, and the final product, trypanothione, from glutathione and spermidine; and an amidase domain, which can hydrolyse glutathionylspermidine and trypanothione to the original substrates. There has only been one previous study that succeeded in resolving these domains from the homologous enzyme glutathionylspermidine synthetase (GspS) from Escherichia coli [6]. GspS typically shares about 48% amino acid similarity and 32% identity with the TryS proteins from the various species. To date, the threedimensional crystal structure for GspS and TryS has not been elucidated and although a short communication outlining the optimized NMR conditions for the N-terminal amidase domain of GspS from E. coli was published a number of years ago [7], no subsequent data have been released. Little is known about the active sites of TryS, although site-directed mutagenesis has highlighted an essential Cys residue for the amidase activity of GspS from Crithidia fasciculata which is conserved across all species of GspS and TryS [8]. In addition, the important roles of Arg-553 and Arg-613 for synthetase activity were recently identified in TryS from C. fasciculata [9]. Our attempts to crystallise the full length TryS from the various species of trypanosomatids have so far been met with limited suc

Biosynthesis of NAD(P) cofactors is of special importance for cyanobacteria due to their role in photosynthesis and respiration. Despite significant progress in understanding NAD(P) biosynthetic machinery in some model organisms, relatively little is known about its implementation in cyanobacteria. We addressed this problem by a combination of comparative genome analysis with verification experiments in the model system ofSynechocystissp. strain PCC 6803. A detailed reconstruction of the NAD(P) metabolic subsystem using the SEED genomic platform (http://theseed.uchicago.edu/FIG/index.cgi) helped us accurately annotate respective genes in the entire set of 13 cyanobacterial species with completely sequenced genomes available at the time. Comparative analysis of operational variants implemented in this divergent group allowed us to elucidate both conserved (de novo and universal pathways) and variable (recycling and salvage pathways) aspects of this subsystem. Focused genetic and biochemical experiments confirmed several conjectures about the key aspects of this subsystem. (i) The product of the slr1691 gene, a homolog ofEscherichia coligenenadEcontaining an additional nitrilase-like N-terminal domain, is a NAD synthetase capable of utilizing glutamine as an amide donor in vitro. (ii) The product of the sll1916 gene, a homolog ofE. coligenenadD, is a nicotinic acid mononucleotide-preferring adenylyltransferase. This gene is essential for survival and cannot be compensated for by an alternative nicotinamide mononucleotide (NMN)-preferring adenylyltransferase (slr0787 gene). (iii) The product of the slr0788 gene is a nicotinamide-preferring phosphoribosyltransferase involved in the first step of the two-step nondeamidating utilization of nicotinamide (NMN shunt). (iv) The physiological role of this pathway encoded by a conserved gene cluster, slr0787-slr0788, is likely in the recycling of endogenously generated nicotinamide, as supported by the inability of this organism to utilize exogenously provided niacin. Positional clustering and the cooccurrence profile of the respective genes across a diverse collection of cellular organisms provide evidence of horizontal transfer events in the evolutionary history of this pathway.

We studied the effects of polyamines, which are necessary for proliferation and antioxidation in Trypanosoma brucei gambiense Wellcome strain (WS) and Trypanosoma brucei brucei ILtat 1.4 strain (IL). No difference was found in activity of ornithine decarboxylase (ODC), a key enzyme in polyamine synthesis in trypanosomes, in both strains maintained in vitro; higher (P0.05) ODC values were found in IL in vivo. However, WS in vivo exhibited higher proliferation rates with higher spermidine content and decreased host survival times than IL. The in vitro proliferation and polyamine contents of WS increased with the addition of polyamine to the 1-difluoromethylornithine culture medium, but not IL. These results suggested that WS uses extracellular polyamine for proliferation. In the in vitro culture, WS was less tolerant of hydrogen peroxide (oxidative stress) than IL, and malondialdehyde levels in WS were higher than in IL. The expression of trypanothione synthetase mRNA in WS in vitro was higher than in IL. These results suggest that IL is dependent on the synthesis of polyamines for proliferation and reduction of oxidative stress, whereas WS is dependent on the uptake of extracellular polyamines. A thorough understanding of the differences in the metabolic capabilities of various trypanosomes is important for the design of more effective medical treatments.

Coronafacic acid, the polyketide component of the phytotoxin coronatine, is activated and coupled to coronamic acid via amide bond formation, a biosynthetic step presumably catalyzed by the coronafacate ligase (cfl) gene product. In the present study, cfl was fused to the carboxy terminus of malE, which encodes the maltose-binding protein (MBP), and overexpressed in Escherichia coli. Immunoblot analysis indicated that Cfl contained an ATP-binding region, a motif conserved in enzymes which activate their substrates by adenylation. MBP-Cfl was overproduced and purified from Pseudomonas syringae and the protein fusion was used to generate antisera. Anti-MBP-Cfl antibodies and a transcriptional fusion of the cfl promoter to a promoterless glucuronidase gene were used to follow the temporal expression of coronafacate ligase. The results indicated that transcription of cfl is temperature-sensitive. Furthermore, a nonpolar mutation in cfl suggested that the gene may have a role in coronafacic acid biosynthesis.

As a part of our project aimed at the search for new safe chemotherapeutic and chemoprophylactic agents against American trypanosomiasis (Chagas's disease); a series of phosphinopeptides structurally related to glutathione was designed, synthesized, and evaluated as antiproliferative agents against the parasite responsible for this disease, the hemoflagellated protozoan Trypanosoma cruzi. The rationale for the synthesis of these compounds was supported on the basis that the presence of the phosphinic acid moiety would mimic the tetrahedral transition state of trypanothione synthase (TryS), a typical C:N ligase, and the molecular target of these drugs. Of the designed compounds, 53 and 54 were potent growth inhibitors against the clinically more relevant form of T. cruzi (amastigotes) growing in myoblasts. The efficacy for these drugs was comparable to that exhibited by the well-known antiparasitic agent WC-9. The simple phosphinopeptide structure found as a pharmacophore in the present study constitutes a starting point for the development of straightforward optimized drugs. © 2006 American Chemical Society. Fil: Ravaschino, Esteban L.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; Argentina Fil: Docampo, Roberta. UniVersity of Georgia; Grecia Fil: Rodriguez, Juan Bautista. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; Argentina

We previously reported that tethered dimers containing indoles on one end and a permanent positive charge on the other, using a 6-9 carbon polymethylene tether, provided NAD synthetase inhibitors with impressive antibacterial activities against Gram-positives. Here, we report that the phenyl ring is a good substitute for indole, and we utilize solution-phase parallel synthesis to explore structure-activity relationships for substituents on that ring. General conclusions are that nonpolar substituents are more effective than polar ones and that different positional isomers often have very different enzyme inhibition activities. This latter observation reveals that enzyme activity is sensitive to minor structural changes and suggests that nonspecific detergent actions are not important for the observed effects.

Trypanothione plays a pivotal role in defence against chemical and oxidant stress, thiol redox homoeostasis, ribonucleotide metabolism and drug resistance in parasitic kinetoplastids. In Trypanosoma brucei, trypanothione is synthesized from glutathione and spermidine by a single enzyme, TryS (trypanothione synthetase), with glutathionylspermidine as an intermediate. To examine the physiological roles of trypanothione, tetracycline-inducible RNA interference was used to reduce expression of TRYS. Following induction, TryS protein was reduced >10-fold and growth rate was reduced 2-fold, with concurrent 5–10-fold decreases in glutathionylspermidine and trypanothione and an up to 14-fold increase in free glutathione content. Polyamine levels were not significantly different from non-induced controls, and neither was the intracellular thiol redox potential, indicating that these factors are not responsible for the growth defect. Compensatory changes in other pathway enzymes were associated with prolonged suppression of TryS: an increase in trypanothione reductase and γ-glutamylcysteine synthetase, and a transient decrease in ornithine decarboxylase. Depleted trypanothione levels were associated with increases in sensitivity to arsenical, antimonial and nitro drugs, implicating trypanothione metabolism in their mode of action. Escape mutants arose after 2 weeks of induction, with all parameters, including growth, returning to normal. Selective inhibitors of TryS are required to fully validate this novel drug target.

NAD synthetase is responsible for the conversion of nicotinic acid adenine dinucleotide to nicotinamide adenine dinucleotide. This reaction provides a biosynthetic route of the coenzyme and, thus, a source of cellular reducing equivalents. Alterations in the oxidative reductive potential of the cell have been implicated as a contributing factor in many disease states. Thus, this enzyme represents a new class of potential drug targets, and, hence, our efforts were focused upon developing a robust assay for utilization in a high throughput screen. Toward that end, we describe a coupled enzyme assay format for the measurement of recombinant human NAD synthetase by employing lactate dehydrogenase in a cycling/amplification reaction linked ultimately to the fluorescence generation of resorufin from resazurin via diaphorase. We present kinetics of the reaction of NAD synthetase in the coupled assay format, optimization conditions, and inhibition of the reaction by gossypol [1,1',6,6',7,7'-hexahydroxy-3,3'-dimethyl-5,5'-bis(1-methylethyl)-[2,2'- binaphthalene]-8,8'-dicarboxaldehyde] and illustrate the robustness of the assay by demonstrating 384-well microtiter plate uniformity statistics. Collectively, our results show that the assay method is both robust and well suited for this class of enzymes involved in the NAD+ biosynthetic pathway.

The determination of theanine has been performed by micellar electrokinetic capillary chromatography (MECC) using 2,4-dinitrofluorobenzene (DNFB) as a derivative reagent. To achieve the separation, a fused-silica capillary column was used with a borax buffer at 0.03 mol/L pH 9.8 (containing Brij35 and isopropanol) at 17 degrees C with detection wave length at 360 nm. The factors affecting the efficiency of the sample separation were examined simultaneously. A 40-min reaction at 35 degrees C between l-glutamate and ethylamine (with Tris-HCl buffer, pH 7.5) was investigated using the theanine synthetase from budding tea seeds. A novel method for the analysis of theanine synthetase activity based on MECC was established. The method shows mean recovery ranged from 87.1 to 105.3% and linearity ranged from 0.2 to 5.0 mmol/L.

The maize Hm1 gene encoding the NADPH-dependent HC-toxin reductase is capable of detoxifying HC-toxin of fungus Cochliobolus carbonum . Here, we conducted the metabolic and biochemical analysis in transgenic rice plants overexpressing an HC-toxin reductase-like gene in rice ( YK1 gene). Methods employing NADPH oxidation and capillary electrophoresis mass spectrometry analysis confirmed that YK1 possessed dihydroflavonol-4-reductase activity in vitro and in vivo . The overexpression of YK1 in both suspension-cultured cells and rice plants increased NAD(H) and NADP(H) levels by causing an increase in NAD synthetase and NAD kinase activities. Activity changes in enzymes that require NAD(P) as coenzymes were also noted in rice cells ectopically expressing YK1, where the cell death caused by hydrogen peroxide and bacterial disease was down-regulated. Thus, a strategy was proposed that the combination of dihydroflavonol-4-reductase activity and the elevated level of NAD(P)H pool may confer the prevention of induced cell death in planta .

Background Ammonia is an important marker for liver diseases such as hepatitis and hepatic cirrhosis. Several methods have been developed for ammonia analysis. In particular, the enzymatic assay using glutamate dehydrogenase has been widely used. However, this method is not necessarily high in sensitivity and accuracy due to inhibition by interferences in plasma and instability over long-term storage. Methods We developed an ammonia assay using a system consisting of three enzymes, NAD synthetase (NADS; EC 6.3.1.5), glucose dehydrogenase (GlcDH; EC 1.1.1.47), and diaphorase (DI; EC 1.6.99.2). Results The calibration curve for ammonia with the cycling method was linear (r=0.999) up to 300 μmol/l. The within-run CVs of 10 and 20 μmol/l NH4Cl solutions and 24.1 μmol/l ammonia in human plasma were 2.3%, 1.5%, and 2.8%, respectively. The between-run CVs of them were 4.5%, 3.1%, and 2.8%, respectively. The recovery was between 96.3% and 105%, and the limit of detection was 2.4 μmol/l. No significant interference was observed with addition of the following components: hemoglobin, bilirubin, chyle, EDTA, heparin, and sodium citrate. Due to the high degree of specificity of NAD synthetase to ammonia, no amino compounds exhibited any effect on the ammonia assay. A high correlation was obtained between results of the present method (y) and a conventional glutamate dehydrogenase method in regression analysis; y=0.944x−6.160 with r=0.993 (n=125). However, an addition error was observed from Bland–Altman analysis (the 95% limits of agreement between the two methods; 9.51±5.92 μmol/l). Conclusion This new enzymatic method is more sensitive, precise, and accurate than the conventional method. In particular, accurate assay for ammonia can be performed without interference in the presence of various compounds.

The accurate description of solvation effects is highly desirable in numerous computational chemistry applications. One widely used methodology treats the solvent as a uniform continuum ("implicit solvation"), and describes its net interaction with the solute by solving the Poisson-Boltzmann (PB) equation using the Boundary Element Method (BEM). These calculations are very time consuming using conventional computers. A new, efficient way of solving the PB equation on the special-purpose hardware chip MDGRAPE-2 is presented. The MDGRAPE-2 chip was originally developed to speed up the calculation of pairwise van der Waals and electrostatic interactions. By recasting the BEM equations and applying an iterative solution procedure, a hardware-accelerated PB solver on MDGRAPE-2 could be implemented. The performance and reliability of the method is demonstrated on three examples ranging from small peptides to large proteins, whereby the obtained acceleration factors range from 15-fold to up to 40-fold with no loss of accuracy compared to the conventional approach.

Acute ammonia toxicity is mediated by excessive activation of NMDA receptors. Activation of NMDA receptors leads to activation of poly(ADP-ribose) polymerase (PARP) which mediates NMDA excitotoxicity. PARP is activated following DNA damage and may lead to cell death via NAD+ and ATP depletion. The aim of the present work was to assess whether acute ammonia intoxication in vivo leads to increased PARP in brain cells nuclei and to altered NAD+ and superoxide metabolism and the contribution of NMDA receptors to these alterations. Acute ammonia intoxication increases PARP content twofold in brain cells nuclei.NAD+ content decreased by 55% in rats injected with ammonia. This was not due to decreased NAD+ synthetase nor increased NAD+ hydrolase activities and would be due to increased NAD+ consumption by PARP. Superoxide radical formation increased by 75% in nuclei of brains of rats injected with ammonia, that also induced protein nitrotyrosylation and DNA damage. Blocking NMDA receptors prevented ammonia-induced PARP, superoxide and nitrotyrosylation increase, DNA damage and NAD+ decrease. These results show that acute ammonia intoxication in vivo leads to activation of NMDA receptors, leading to increased superoxide formation and PARP content and depletion of NAD+ in brain cells nuclei that contribute to ammonia toxicity.

The aminocoumarin antibiotics clorobiocin, novobiocin, and coumermycin A 1 are inhibitors of bacterial gyrase. Their chemical structures contain amide bonds, formed between an aminocoumarin ring and an aromatic acyl component, which is 3-dimethylallyl-4-hydroxybenzoate in the case of novobiocin and clorobiocin. These amide bonds are formed under catalysis of the gene products of cloL , novL , and couL , respectively. We first examined the substrate specificity of the purified amide synthetases CloL, NovL, and CouL for the various analogs of the prenylated benzoate moiety. We then generated new aminocoumarin antibiotics by feeding synthetic analogs of the 3-dimethylallyl-4-hydroxybenzoate moiety to a mutant strain defective in the biosynthesis of the prenylated benzoate moiety. This resulted in the formation of 32 new aminocoumarin compounds. The structures of these compounds were elucidated using FAB-MS and 1 H-NMR spectroscopy.

The production of nitric oxide and other reactive nitrogen intermediates (RNI) by macrophages helps to control infection byMycobacterium tuberculosis(Mtb). However, the protection is imperfect and infection persists. To identify genes thatMtbrequires to resist RNI, we screened 10,100Mtbtransposon mutants for hypersusceptibility to acidified nitrite. We found 12 mutants with insertions in seven genes representing six pathways, including the repair of DNA (uvrB) and the synthesis of a flavin cofactor (fbiC). Five mutants had insertions in proteasome-associated genes. AnMtbmutant deficient in a presumptive proteasomal adenosine triphosphatase was attenuated in mice, and exposure to proteasomal protease inhibitors markedly sensitized wild-typeMtbto RNI. Thus, the mycobacterial proteasome serves as a defense against oxidative or nitrosative stress.

Nicotinamide-adenine dinucleotide (NAD+) synthetases catalyze the last step in NAD+ metabolism in the de novo, import, and salvage pathways that originate from tryptophan (or aspartic acid), nicotinic acid, and nicotinamide, respectively, and converge on nicotinic acid mononucleotide. NAD+ synthetase converts nicotinic acid adenine dinucleotide to NAD+ via an adenylylated intermediate. All of the known eukaryotic NAD+ synthetases are glutamine-dependent, hydrolyzing glutamine to glutamic acid to provide the attacking ammonia. In the prokaryotic world, some NAD+ synthetases are glutamine-dependent, whereas others can only use ammonia. Earlier, we noted a perfect correlation between presence of a domain related to nitrilase and glutamine dependence and then proved in the accompanying paper (Bieganowski, P., Pace, H. C., and Brenner, C. (2003) J. Biol. Chem. 278, 33049-33055) that the nitrilase-related domain is an essential, obligate intramolecular, thiol-dependent glutamine amidotransferase in the yeast NAD+ synthetase, Qns1. Independently, human NAD+ synthetase was cloned and shown to depend on Cys-175 for glutamine-dependent but not ammonia-dependent NAD+ synthetase activity. Additionally, it was claimed that a 275 amino acid open reading frame putatively amplified from human glioma cell line LN229 encodes a human ammonia-dependent NAD+ synthetase and this was speculated largely to mediate NAD+ synthesis in human muscle tissues. Here we establish that the so-called NADsyn2 is simply ammonia-dependent NAD+ synthetase from Pseudomonas, which is encoded on an operon with nicotinic acid phosphoribosyltransferase and, in some Pseudomonads, with nicotinamidase.

Seven carbapenem-nonsusceptible Morganella morganii isolates, which have similar antibiotic susceptibility profiles, were isolated over a 5-month period. MICs of imipenem, meropenem, and ertapenem were 8, 1, and 0.25 to 0.5 μg/mL, respectively. Pulsed-field gel electrophoresis indicated that 6 isolates were indistinguishable or closely related. Carbapenem resistance can be transferred from M. morganii to Escherichia coli by conjugation. All M. morganii isolates and E. coli transconjugants produced KPC-2 and carried the qnrS1 gene. Production of KPC-2 mainly contributed to the carbapenem resistance in M. morganii . KPC-2–producing M. morganii clonally spread in a hospital in China.

The structural analysis of all enzymes in a metabolic pathway is a prerequisite to answering fascinating questions, such as those relating to the evolutionary relationships between enzymes within the same and related pathways. Furthermore, the observed impressive diversity of catalytic functions displayed by these enzymes can lead to the synthesis of highly complex or unstable molecules, frequently involving unusual chemical reactions. Moreover, a detailed description of the active site of each enzyme in a pathway is of immense importance for the rational design of new drugs. The recent progress made in the structural biology of enzymes involved in NAD and molybdenum cofactor biosynthesis presents a significant step toward these goals.

The final step of NAD+ biosynthesis includes an amide transfer to nicotinic acid adenine dinucleotide (NaAD) catalyzed by NAD+ synthetase. This enzyme was co-crystallized in microgravity with natural substrates NaAD and ATP at pH 8.5. The crystal was exposed to ammonium ions, synchrotron diffraction data were collected and the atomic model was refined anisotropically at 1 A resolution to R = 11.63%. Both binding sites are occupied by the NAD-adenylate intermediate, pyrophosphate and two magnesium ions. The atomic resolution of the structure allows better definition of non-planar peptide groups, reveals a low mean anisotropy of protein and substrate atoms and indicates the H-­atom positions of the phosphoester group of the reaction intermediate. The phosphoester group is protonated at the carbonyl O atom O7N, suggesting a carbenium-ion structure stabilized by interactions with two solvent sites presumably occupied by ammonia and a water molecule. A mechanism is proposed for the second catalytic step, which includes a nucleophilic attack by the ammonia molecule on the intermediate.

Bacillus stearothermophilus H-804 isolated from a hot spring in Beppu, Japan, produced an ammonia-specific NAD synthetase (EC 6.3.1.5). The enzyme specifically used NH3 as an amide donor for the synthesis of NAD as it formed AMP and pyrophosphate from deamide-NAD and ATP. None of the l-amino acids tested, such as l-asparagine or l-glutamine, or other amino compounds such as urea, uric acid, or creatinine was used instead of NH3. Mg2+ was needed for the activity, and the maximum enzyme activity was obtained with 3 mM MgCl2. The molecular mass of the native enzyme was 50 kDa by gel filtration, and SDS-PAGE showed a single protein band at the molecular mass of 25 kDa. The optimum pH and temperature for the activity were from 9.0 to 10.0 and 60 degrees C, respectively. The enzyme was stable at a pH range of 7.5 to 9.0 and up to 60 degrees C. The Km for NH3, ATP, and deamide-NAD were 0.91, 0.052, and 0.028 mM, respectively. The gene encoding the enzyme consisted of an open reading frame of 738 bp and encoded a protein of 246 amino acid residues. The deduced amino acid sequence of the gene had about 32% homology to those of Escherichia coli and Bacillus subtilis NAD synthetases. We caused the NAD synthetase gene to be expressed in E. coli at a high level; the enzyme activity (per liter of medium) produced by the recombinant E. coli was 180-fold that of B. stearothermophilus H-804. The specific assay of ammonia and ATP (up to 25 microM) with this stable NAD synthetase was possible.

Enzymes of glutamate and GABA metabolism in postmortem cerebellum from patients with Alzheimer's disease (AD) have not been comprehensively studied. The present work reports results of original comparative study on levels of phosphate-activated glutaminase (PAG) and glutamic acid decarboxylase isoenzymes (GAD65/67) in autopsied cerebellum samples from AD patients and matched controls (13 cases in each group) as well as summarizes published evidence for altered levels of PAG and GAD65/67 in AD brain. Altered (decreased) levels of these enzymes and changes in links between amounts of these enzymes and other glutamate-metabolizing enzymes (such as glutamate dehydrogenase and glutamine synthetase-like protein) in AD cerebella suggest significantly impaired glutamate and GABA metabolism in this brain region, which was previously regarded as not substantially involved in AD pathogenesis.