Identification of diphenyl furan derivatives via high throughput and computational studies as ArgA inhibitors of Mycobacterium tuberculosis.

Microbial amino acid biosynthetic pathways are underexploited for the development of anti-bacterial agents. N-acetyl glutamate synthase (ArgA) catalyses the first committed step in L-arginine biosynthesis and is essential for M. tuberculosis growth. Here, we have purified and optimized assay conditions for the acetylation of l-glutamine by ArgA. Using the optimized conditions, high throughput screening was performed to identify ArgA inhibitors. We identified 2,5-Bis (2-chloro-4-guanidinophenyl) furan, a dicationic diaryl furan derivatives, as ArgA inhibitor, with a MIC99 values of 1.56 µM against M. tuberculosis. The diaryl furan derivative displayed bactericidal killing against both M. bovis BCG and M. tuberculosis. Inhibition of ArgA by the lead compound resulted in transcriptional reprogramming and accumulation of reactive oxygen species. The lead compound and its derivatives showed micromolar binding with ArgA as observed in surface plasmon resonance and tryptophan quenching experiments. Computational and dynamic analysis revealed that these scaffolds share similar binding site residues with L-arginine, however, with slight variations in their interaction pattern. Partial restoration of growth upon supplementation of liquid cultures with either L-arginine or N-acetyl cysteine suggests a multi-target killing mechanism for the lead compound. Taken together, we have identified small molecule inhibitors against ArgA enzyme from M. tuberculosis.

Noncoding sequence variants define a novel regulatory element in the first intron of the N-acetylglutamate synthase gene.

N-acetylglutamate synthase deficiency is an autosomal recessive urea cycle disorder caused either by decreased expression of the NAGS gene or defective NAGS enzyme resulting in decreased production of N-acetylglutamate (NAG), an allosteric activator of carbamylphosphate synthetase 1 (CPS1). NAGSD is the only urea cycle disorder that can be effectively treated with a single drug, N-carbamylglutamate (NCG), a stable NAG analog, which activates CPS1 to restore ureagenesis. We describe three patients with NAGSD due to four novel noncoding sequence variants in the NAGS regulatory regions. All three patients had hyperammonemia that resolved upon treatment with NCG. Sequence variants NM_153006.2:c.427-222G>A and NM_153006.2:c.427-218A>C reside in the 547 bp-long first intron of NAGS and define a novel NAGS regulatory element that binds retinoic X receptor α. Sequence variants NC_000017.10:g.42078967A>T (NM_153006.2:c.-3065A>T) and NC_000017.10:g.42078934C>T (NM_153006.2:c.-3098C>T) reside in the NAGS enhancer, within known HNF1 and predicted glucocorticoid receptor binding sites, respectively. Reporter gene assays in HepG2 and HuH-7 cells demonstrated that all four substitutions could result in reduced expression of NAGS. These findings show that analyzing noncoding regions of NAGS and other urea cycle genes can reveal molecular causes of disease and identify novel regulators of ureagenesis.

Clinical and structural insights into potential dominant negative triggers of proximal urea cycle disorders.

Despite biochemical and genetic testing being the golden standards for identification of proximal urea cycle disorders (UCDs), genotype-phenotype correlations are often unclear. Co-occurring partial defects affecting more than one gene have not been demonstrated so far in proximal UCDs. Here, we analyzed the mutational spectrum of 557 suspected proximal UCD individuals. We probed oligomerizing forms of NAGS, CPS1 and OTC, and evaluated the surface exposure of residues mutated in heterozygously affected individuals. BN-PAGE and gel-filtration chromatography were employed to discover protein-protein interactions within recombinant enzymes. From a total of 281 confirmed patients, only 15 were identified as "heterozygous-only" candidates (i.e. single defective allele). Within these cases, the only missense variants to potentially qualify as dominant negative triggers were CPS1 p.Gly401Arg and NAGS p.Thr181Ala and p.Tyr512Cys, as assessed by residue oligomerization capacity and surface exposure. However, all three candidates seem to participate in critical intramolecular functions, thus, unlikely to facilitate protein-protein interactions. This interpretation is further supported by BN-PAGE and gel-filtration analyses revealing no multiprotein proximal urea cycle complex formation. Collectively, genetic analysis, structural considerations and in vitro experiments point against a prominent role of dominant negative effects in human proximal UCDs.

Computer Modeling of N-Acetylglutamate Synthase: From Primary Structure to Elemental Stages of Catalysis.

Three-dimensional full-atom model of the enzyme complex with acetyl-CoA and substrate was constructed on the basis of the primary sequence of amino acid residues of N-acetyl glutamate synthase. Bioinformatics approaches of computer modeling were applied, including multiple sequence alignment, prediction of co-evolutionary contacts, and ab initio folding. On the basis of the results of calculations by classical molecular dynamics and combined quantum and molecular mechanics (QM/MM) methods, the structure of the active site and the reaction mechanism of N-acetylglutamate formation are described. Agreement of the structures of the enzyme-product complexes obtained in computer modeling and in the X-ray studies validates the reliability of modeling predictions.

Effect of arginine on oligomerization and stability of N-acetylglutamate synthase.

N-acetylglutamate synthase (NAGS; E.C.2.3.1.1) catalyzes the formation of N-acetylglutamate (NAG) from acetyl coenzyme A and glutamate. In microorganisms and plants, NAG is the first intermediate of the L-arginine biosynthesis; in animals, NAG is an allosteric activator of carbamylphosphate synthetase I and III. In some bacteria bifunctional N-acetylglutamate synthase-kinase (NAGS-K) catalyzes the first two steps of L-arginine biosynthesis. L-arginine inhibits NAGS in bacteria, fungi, and plants and activates NAGS in mammals. L-arginine increased thermal stability of the NAGS-K from Maricaulis maris (MmNAGS-K) while it destabilized the NAGS-K from Xanthomonas campestris (XcNAGS-K). Analytical gel chromatography and ultracentrifugation indicated tetrameric structure of the MmMNAGS-K in the presence and absence of L-arginine and a tetramer-octamer equilibrium that shifted towards tetramers upon binding of L-arginine for the XcNAGS-K. Analytical gel chromatography of mouse NAGS (mNAGS) indicated either different oligomerization states that are in moderate to slow exchange with each other or deviation from the spherical shape of the mNAGS protein. The partition coefficient of the mNAGS increased in the presence of L-arginine suggesting smaller hydrodynamic radius due to change in either conformation or oligomerization. Different effects of L-arginine on oligomerization of NAGS may have implications for efforts to determine the three-dimensional structure of mammalian NAGS.

Understanding N-Acetyl-L-Glutamate Synthase Deficiency: Mutational Spectrum, Impact of Clinical Mutations on Enzyme Functionality, and Structural Considerations.

N-acetyl-L-glutamate synthase (NAGS) deficiency (NAGSD), the rarest urea cycle defect, is clinically indistinguishable from carbamoyl phosphate synthetase 1 deficiency, rendering the identification of NAGS gene mutations key for differentiation, which is crucial, as only NAGSD has substitutive therapy. Over the last 13 years, we have identified 43 patients from 33 families with NAGS mutations, of which 14 were novel. Overall, 36 NAGS mutations have been found so far in 56 patients from 42 families, of which 76% are homozygous for the mutant allele. 61% of mutations are missense changes. Lack or decrease of NAGS protein is predicted for ∼1/3 of mutations. Missense mutations frequency is inhomogeneous along NAGS: null for exon 1, but six in exon 6, which reflects the paramount substrate binding/catalytic role of the C-terminal domain (GNAT domain). Correspondingly, phenotypes associated with missense mutations mapping in the GNAT domain are more severe than phenotypes of amino acid kinase domain-mapping missense mutations. Enzyme activity and stability assays with 12 mutations introduced into pure recombinant Pseudomonas aeruginosa NAGS, together with in silico structural analysis, support the pathogenic role of most NAGSD-associated mutations found. The disease-causing mechanisms appear to be, from higher to lower frequency, decreased solubility/stability, aberrant kinetics/catalysis, and altered arginine modulation.

Metabolic Regulation of Histone Acetyltransferases by Endogenous Acyl-CoA Cofactors.

The finding that chromatin modifications are sensitive to changes in cellular cofactor levels potentially links altered tumor cell metabolism and gene expression. However, the specific enzymes and metabolites that connect these two processes remain obscure. Characterizing these metabolic-epigenetic axes is critical to understanding how metabolism supports signaling in cancer, and developing therapeutic strategies to disrupt this process. Here, we describe a chemical approach to define the metabolic regulation of lysine acetyltransferase (KAT) enzymes. Using a novel chemoproteomic probe, we identify a previously unreported interaction between palmitoyl coenzyme A (palmitoyl-CoA) and KAT enzymes. Further analysis reveals that palmitoyl-CoA is a potent inhibitor of KAT activity and that fatty acyl-CoA precursors reduce cellular histone acetylation levels. These studies implicate fatty acyl-CoAs as endogenous regulators of histone acetylation, and suggest novel strategies for the investigation and metabolic modulation of epigenetic signaling.

The N-Acetylglutamate Synthase Family: Structures, Function and Mechanisms.

N-acetylglutamate synthase (NAGS) catalyzes the production of N-acetylglutamate (NAG) from acetyl-CoA and L-glutamate. In microorganisms and plants, the enzyme functions in the arginine biosynthetic pathway, while in mammals, its major role is to produce the essential co-factor of carbamoyl phosphate synthetase 1 (CPS1) in the urea cycle. Recent work has shown that several different genes encode enzymes that can catalyze NAG formation. A bifunctional enzyme was identified in certain bacteria, which catalyzes both NAGS and N-acetylglutamate kinase (NAGK) activities, the first two steps of the arginine biosynthetic pathway. Interestingly, these bifunctional enzymes have higher sequence similarity to vertebrate NAGS than those of the classical (mono-functional) bacterial NAGS. Solving the structures for both classical bacterial NAGS and bifunctional vertebrate-like NAGS/K has advanced our insight into the regulation and catalytic mechanisms of NAGS, and the evolutionary relationship between the two NAGS groups.

Development of stable isotope dilution assays for the quantitation of Amadori compounds in foods.

During thermal processing of foods, reducing carbohydrates and amino acids may form 1-amino-1-desoxyketoses named Amadori rearrangement products after the Italian chemist Mario Amadori. Although these compounds are transient intermediates of the Maillard reaction, they are often used as suitable markers to measure the extent of a thermal food processing, such as for spray-dried milk or dried fruits. Several methods are already available in the literature for their quantitation, but measurements are often done with external calibration without addressing losses during the workup procedure. To cope with this challenge, stable isotope dilution assays in combination with LC-MS/MS were developed for the glucose-derived Amadori products of the seven amino acids valine, leucine, isoleucine, phenylalanine, tyrosine, methionine, and histidine using the respective synthesized [(13)C6]-labeled isotopologues as internal standards. The quantitation of the analytes added to a model matrix showed a very good sensitivity with the lowest limits of detection for the Amadori compound of phenylalanine of 0.1 µg/kg starch and 0.2 µg/kg oil, respectively. Also, the standard deviation measured in, for example, wheat beer was only ±2% for this analyte. Application of the method to several foods showed the highest concentrations of the Amadori product of valine in unroasted cocoa (342 mg/kg) as well as in dried bell pepper (3460 mg/kg). In agreement with literature data, drying of foods led to the formation of Amadori products, whereas they were degraded during roasting of, for example, coffee or cocoa. The study presents for the first time results on concentrations of the Amadori compounds of tyrosine and histidine in foods.

Expression pattern and biochemical properties of zebrafish N-acetylglutamate synthase.

The urea cycle converts ammonia, a waste product of protein catabolism, into urea. Because fish dispose ammonia directly into water, the role of the urea cycle in fish remains unknown. Six enzymes, N-acetylglutamate synthase (NAGS), carbamylphosphate synthetase III, ornithine transcarbamylase, argininosuccinate synthase, argininosuccinate lyase and arginase 1, and two membrane transporters, ornithine transporter and aralar, comprise the urea cycle. The genes for all six enzymes and both transporters are present in the zebrafish genome. NAGS (EC 2.3.1.1) catalyzes the formation of N-acetylglutamate from glutamate and acetyl coenzyme A and in zebrafish is partially inhibited by L-arginine. NAGS and other urea cycle genes are highly expressed during the first four days of zebrafish development. Sequence alignment of NAGS proteins from six fish species revealed three regions of sequence conservation: the mitochondrial targeting signal (MTS) at the N-terminus, followed by the variable and conserved segments. Removal of the MTS yields mature zebrafish NAGS (zfNAGS-M) while removal of the variable segment from zfNAGS-M results in conserved NAGS (zfNAGS-C). Both zfNAGS-M and zfNAGS-C are tetramers in the absence of L-arginine; addition of L-arginine decreased partition coefficients of both proteins. The zfNAGS-C unfolds over a broader temperature range and has higher specific activity than zfNAGS-M. In the presence of L-arginine the apparent Vmax of zfNAGS-M and zfNAGS-C decreased, their Km(app) for acetyl coenzyme A increased while the Km(app) for glutamate remained unchanged. The expression pattern of NAGS and other urea cycle genes in developing zebrafish suggests that they may have a role in citrulline and/or arginine biosynthesis during the first day of development and in ammonia detoxification thereafter. Biophysical and biochemical properties of zebrafish NAGS suggest that the variable segment may stabilize a tetrameric state of zfNAGS-M and that under physiological conditions zebrafish NAGS catalyzes formation of N-acetylglutamate at the maximal rate.

Crystal structure of the N-acetyltransferase domain of human N-acetyl-L-glutamate synthase in complex with N-acetyl-L-glutamate provides insights into its catalytic and regulatory mechanisms.

N-acetylglutamate synthase (NAGS) catalyzes the conversion of AcCoA and L-glutamate to CoA and N-acetyl-L-glutamate (NAG), an obligate cofactor for carbamyl phosphate synthetase I (CPSI) in the urea cycle. NAGS deficiency results in elevated levels of plasma ammonia which is neurotoxic. We report herein the first crystal structure of human NAGS, that of the catalytic N-acetyltransferase (hNAT) domain with N-acetyl-L-glutamate bound at 2.1 Å resolution. Functional studies indicate that the hNAT domain retains catalytic activity in the absence of the amino acid kinase (AAK) domain. Instead, the major functions of the AAK domain appear to be providing a binding site for the allosteric activator, L-arginine, and an N-terminal proline-rich motif that is likely to function in signal transduction to CPS1. Crystalline hNAT forms a dimer similar to the NAT-NAT dimers that form in crystals of bifunctional N-acetylglutamate synthase/kinase (NAGS/K) from Maricaulis maris and also exists as a dimer in solution. The structure of the NAG binding site, in combination with mutagenesis studies, provide insights into the catalytic mechanism. We also show that native NAGS from human and mouse exists in tetrameric form, similar to those of bifunctional NAGS/K.

Structure of N-acetyl-L-glutamate synthase/kinase from Maricaulis maris with the allosteric inhibitor L-arginine bound.

Maricaulis maris N-acetylglutamate synthase/kinase (mmNAGS/K) catalyzes the first two steps in L-arginine biosynthesis and has a high degree of sequence and structural homology to human N-acetylglutamate synthase, a regulator of the urea cycle. The synthase activity of both mmNAGS/K and human NAGS are regulated by L-arginine, although L-arginine is an allosteric inhibitor of mmNAGS/K, but an activator of human NAGS. To investigate the mechanism of allosteric inhibition of mmNAGS/K by L-arginine, we have determined the structure of the mmNAGS/K complexed with L-arginine at 2.8 Å resolution. In contrast to the structure of mmNAGS/K in the absence of L-arginine where there are conformational differences between the four subunits in the asymmetric unit, all four subunits in the L-arginine liganded structure have very similar conformations. In this conformation, the AcCoA binding site in the N-acetyltransferase (NAT) domain is blocked by a loop from the amino acid kinase (AAK) domain, as a result of a domain rotation that occurs when L-arginine binds. This structural change provides an explanation for the allosteric inhibition of mmNAGS/K and related enzymes by L-arginine. The allosterically regulated mechanism for mmNAGS/K differs significantly from that for Neisseria gonorrhoeae NAGS (ngNAGS). To define the active site, several residues near the putative active site were mutated and their activities determined. These experiments identify roles for Lys356, Arg386, Asn391 and Tyr397 in the catalytic mechanism.

Biochemical characterization of Hpa2 and Hpa3, two small closely related acetyltransferases from Saccharomyces cerevisiae.

Based on their sequences, the Saccharomyces cerevisiae Hpa2 and Hpa3 proteins are annotated as two closely related members of the Gcn5 acetyltransferase family. Here, we describe the biochemical characterization of Hpa2 and Hpa3 as bona fide acetyltransferases with different substrate specificities. Mutational and MALDI-TOF analyses showed that Hpa3 translation initiates primarily from Met-19 rather than the annotated start site, Met-1, with a minor product starting at Met-27. When expressed in Escherichia coli and assayed in vitro, Hpa2 and Hpa3 (from Met-19) acetylated histones and polyamines. Whereas Hpa2 acetylated histones H3 and H4 (at H3 Lys-14, H4 Lys-5, and H4 Lys-12), Hpa3 acetylated only histone H4 (at Lys-8). Additionally, Hpa2, but not Hpa3, acetylated certain small basic proteins. Hpa3, but not Hpa2, has been reported to acetylate D-amino acids, and we present results consistent with that. Overexpression of Hpa2 or Hpa3 is toxic to yeast cells. However, their deletions do not show any standard phenotypic defects. These results suggest that Hpa2 and Hpa3 are similar but distinct acetyltransferases that might have overlapping roles with other known acetyltransferases in vivo in acetylating histones and other small proteins.

Structure of the complex of Neisseria gonorrhoeae N-acetyl-L-glutamate synthase with a bound bisubstrate analog.

N-Acetyl-L-glutamate synthase catalyzes the conversion of AcCoA and glutamate to CoA and N-acetyl-L-glutamate (NAG), the first step of the arginine biosynthetic pathway in lower organisms. In mammals, NAG is an obligate cofactor of carbamoyl phosphate synthetase I in the urea cycle. We have previously reported the structures of NAGS from Neisseria gonorrhoeae (ngNAGS) with various substrates bound. Here we reported the preparation of the bisubstrate analog, CoA-S-acetyl-L-glutamate, the crystal structure of ngNAGS with CoA-NAG bound, and kinetic studies of several active site mutants. The results are consistent with a one-step nucleophilic addition-elimination mechanism with Glu353 as the catalytic base and Ser392 as the catalytic acid. The structure of the ngNAGS-bisubstrate complex together with the previous ngNAGS structures delineates the catalytic reaction path for ngNAGS.

Purification of chicken breast protein hydrolysate and analysis of its antioxidant activity.

Chicken breast protein was hydrolyzed by papain under optimal conditions. The antioxidant activity of the chicken breast protein hydrolysate was then evaluated in vitro and in vivo using different measurements, including reducing power and DPPH radical scavenging assays. The reducing power of the hydrolysate was 0.5 at 2.37 mg/mL. The DPPH radical scavenging assay showed that the EC50 value of the hydrolysate was 1.28 mg/mL. In antioxidant assays in vivo, d-galactose-induced aging mice administrated the fraction peptides of chicken breast protein hydrolysate showed significantly increased antioxidant enzyme activities, while malondialdehyde levels decreased both in serums and livers. Under a transmission electron microscope (TEM), the ultramicrostructure of hepatic tissue was observed and we found that the hydrolysate may play a part in inhibiting oxidative stress in hepatocytes in vivo. Therefore, we concluded that chicken breast protein hydrolysate exhibits significant antioxidant activity.

Insight on an arginine synthesis metabolon from the tetrameric structure of yeast acetylglutamate kinase.

N-acetyl-L-glutamate kinase (NAGK) catalyzes the second, generally controlling, step of arginine biosynthesis. In yeasts, NAGK exists either alone or forming a metabolon with N-acetyl-L-glutamate synthase (NAGS), which catalyzes the first step and exists only within the metabolon. Yeast NAGK (yNAGK) has, in addition to the amino acid kinase (AAK) domain found in other NAGKs, a ~150-residue C-terminal domain of unclear significance belonging to the DUF619 domain family. We deleted this domain, proving that it stabilizes yNAGK, slows catalysis and modulates feed-back inhibition by arginine. We determined the crystal structures of both the DUF619 domain-lacking yNAGK, ligand-free as well as complexed with acetylglutamate or acetylglutamate and arginine, and of complete mature yNAGK. While all other known arginine-inhibitable NAGKs are doughnut-like hexameric trimers of dimers of AAK domains, yNAGK has as central structure a flat tetramer formed by two dimers of AAK domains. These dimers differ from canonical AAK dimers in the -110° rotation of one subunit with respect to the other. In the hexameric enzymes, an N-terminal extension, found in all arginine-inhibitable NAGKs, forms a protruding helix that interlaces the dimers. In yNAGK, however, it conforms a two-helix platform that mediates interdimeric interactions. Arginine appears to freeze an open inactive AAK domain conformation. In the complete yNAGK structure, two pairs of DUF619 domains flank the AAK domain tetramer, providing a mechanism for the DUF619 domain modulatory functions. The DUF619 domain exhibits the histone acetyltransferase fold, resembling the catalytic domain of bacterial NAGS. However, the putative acetyl CoA site is blocked, explaining the lack of NAGS activity of yNAGK. We conclude that the tetrameric architecture is an adaptation to metabolon formation and propose an organization for this metabolon, suggesting that yNAGK may be a good model also for yeast and human NAGSs.

Functional dissection of N-acetylglutamate synthase (ArgA) of Pseudomonas aeruginosa and restoration of its ancestral N-acetylglutamate kinase activity.

In many microorganisms, the first step of arginine biosynthesis is catalyzed by the classical N-acetylglutamate synthase (NAGS), an enzyme composed of N-terminal amino acid kinase (AAK) and C-terminal histone acetyltransferase (GNAT) domains that bind the feedback inhibitor arginine and the substrates, respectively. In NAGS, three AAK domain dimers are interlinked by their N-terminal helices, conforming a hexameric ring, whereas each GNAT domain sits on the AAK domain of an adjacent dimer. The arginine inhibition of Pseudomonas aeruginosa NAGS was strongly hampered, abolished, or even reverted to modest activation by changes in the length/sequence of the short linker connecting both domains, supporting a crucial role of this linker in arginine regulation. Linker cleavage or recombinant domain production allowed the isolation of each NAGS domain. The AAK domain was hexameric and inactive, whereas the GNAT domain was monomeric/dimeric and catalytically active although with ∼50-fold-increased and ∼3-fold-decreased K(m)(glutamate) and k(cat) values, respectively, with arginine not influencing its activity. The deletion of N-terminal residues 1 to 12 dissociated NAGS into active dimers, catalyzing the reaction with substrate kinetics and arginine insensitivity identical to those for the GNAT domain. Therefore, the interaction between the AAK and GNAT domains from different dimers modulates GNAT domain activity, whereas the hexameric architecture appears to be essential for arginine inhibition. We proved the closeness of the AAK domains of NAGS and N-acetylglutamate kinase (NAGK), the enzyme that catalyzes the next arginine biosynthesis step, shedding light on the origin of classical NAGS, by showing that a double mutation (M26K L240K) in the isolated NAGS AAK domain elicited NAGK activity.

A novel N-acetylglutamate synthase architecture revealed by the crystal structure of the bifunctional enzyme from Maricaulis maris.

Novel bifunctional N-acetylglutamate synthase/kinases (NAGS/K) that catalyze the first two steps of arginine biosynthesis and are homologous to vertebrate N-acetylglutamate synthase (NAGS), an essential cofactor-producing enzyme in the urea cycle, were identified in Maricaulis maris and several other bacteria. Arginine is an allosteric inhibitor of NAGS but not NAGK activity. The crystal structure of M. maris NAGS/K (mmNAGS/K) at 2.7 Å resolution indicates that it is a tetramer, in contrast to the hexameric structure of Neisseria gonorrhoeae NAGS. The quaternary structure of crystalline NAGS/K from Xanthomonas campestris (xcNAGS/K) is similar, and cross-linking experiments indicate that both mmNAGS/K and xcNAGS are tetramers in solution. Each subunit has an amino acid kinase (AAK) domain, which is likely responsible for N-acetylglutamate kinase (NAGK) activity and has a putative arginine binding site, and an N-acetyltransferase (NAT) domain that contains the putative NAGS active site. These structures and sequence comparisons suggest that the linker residue 291 may determine whether arginine acts as an allosteric inhibitor or activator in homologous enzymes in microorganisms and vertebrates. In addition, the angle of rotation between AAK and NAT domains varies among crystal forms and subunits within the tetramer. A rotation of 26° is sufficient to close the predicted AcCoA binding site, thus reducing enzymatic activity. Since mmNAGS/K has the highest degree of sequence homology to vertebrate NAGS of NAGS and NAGK enzymes whose structures have been determined, the mmNAGS/K structure was used to develop a structural model of human NAGS that is fully consistent with the functional effects of the 14 missense mutations that were identified in NAGS-deficient patients.

Biochemical and thermodynamic analyses of Salmonella enterica Pat, a multidomain, multimeric N(ε)-lysine acetyltransferase involved in carbon and energy metabolism.

In the bacterium Salmonella enterica, the CobB sirtuin protein deacetylase and the Gcn5-related N(ε)-acetyltransferase (GNAT) Pat control carbon utilization and metabolic flux via N(ε)-lysine acetylation/deacetylation of metabolic enzymes. To date, the S. enterica Pat (SePat) acetyltransferase has not been biochemically characterized. Here we report the kinetic and thermodynamic characterization of the SePat enzyme using two of its substrates, acetyl coenzyme A (Ac-CoA) synthetase (Acs; AMP forming, EC 6.2.1.1) and Ac-CoA. The data showed typical Michaelis-Menten kinetic behavior when Ac-CoA was held at a saturating concentration while Acs was varied, and a sigmoidal kinetic behavior was observed when Acs was saturating and the Ac-CoA concentration was varied. The observation of sigmoidal kinetics and positive cooperativity for Ac-CoA is an unusual feature of GNATs. Results of isothermal titration calorimetry (ITC) experiments showed that binding of Ac-CoA to wild-type SePat produced a biphasic curve having thermodynamic properties consistent with two distinct sites. Biphasicity was not observed in ITC experiments that analyzed the binding of Ac-CoA to a C-terminal construct of SePat encompassing the predicted core acetyltransferase domain. Subsequent analytical gel filtration chromatography studies showed that in the presence of Ac-CoA, SePat oligomerized to a tetrameric form, whereas in the absence of Ac-CoA, SePat behaved as a monomer. The positive modulation of SePat activity by Ac-CoA, a product of the Acs enzyme that also serves as a substrate for SePat-dependent acetylation, is likely a layer of metabolic control. IMPORTANCE For decades, N(ε)-lysine acetylation has been a well-studied mode of regulation of diverse proteins involved in almost all aspects of eukaryotic physiology. Until recently, N(ε)-lysine acetylation was not considered a widespread phenomenon in bacteria. Recent studies have indicated that N(ε)-lysine acetylation and its impact on cellular metabolism may be just as diverse in bacteria as they are in eukaryotes. The S. enterica Pat enzyme, specifically, has recently been implicated in the modulation of many metabolic enzymes. Understanding the molecular mechanisms of how this enzyme controls the activity of diverse enzymes by N(ε)-lysine acetylation will advance our understanding of how the prokaryotic cell responds to its changing environment in order to meet its metabolic needs.

N-acetylglutamate synthase: structure, function and defects.

N-acetylglutamate (NAG) is a unique enzyme cofactor, essential for liver ureagenesis in mammals while it is the first committed substrate for de novo arginine biosynthesis in microorganisms and plants. The enzyme that produces NAG from glutamate and CoA, NAG synthase (NAGS), is allosterically inhibited by arginine in microorganisms and plants and activated in mammals. This transition of the allosteric effect occurred when tetrapods moved from sea to land. The first mammalian NAGS gene (from mouse) was cloned in 2002 and revealed significant differences from the NAGS ortholog in microorganisms. Almost all NAGS genes possess a C-terminus transferase domain in which the catalytic activity resides and an N-terminus kinase domain where arginine binds. The three-dimensional structure of NAGS shows two distinctly folded domains. The kinase domain binds arginine while the acetyltransferase domain contains the catalytic site. NAGS deficiency in humans leads to hyperammonemia and can be primary, due to mutations in the NAGS gene or secondary due to other mitochondrial aberrations that interfere with the normal function of the same enzyme. For either condition, N-carbamylglutamate (NCG), a stable functional analog of NAG, was found to either restore or improve the deficient urea-cycle function.