Step Of Arginine Biosynthesis Research Articles

Argininosuccinate Synthetase-1 (ASS1) Loss in High-Grade Neuroendocrine Carcinomas of the Urinary Bladder: Implications for Targeted Therapy with ADI-PEG 20.

High-grade neuroendocrine carcinomas (HGNECs) of the urinary bladder encompass small cell (SCNEC) and large cell neuroendocrine carcinomas (LCNEC). Currently, recommended initial management is with systemic chemotherapy, followed by consolidative therapy with either radical cystectomy or radiotherapy in patients with localized disease. Nevertheless, survival in this setting remains poor. We therefore evaluated the potential to modify arginine metabolism as an alternative, targeted therapy approach in these carcinomas. In humans, arginine is a semi-essential amino acid and its synthesis enzyme argininosuccinate synthetase (ASS1) represents the rate-limiting step in arginine biosynthesis. Neoplasms that show low to absent ASS1 expression require extracellular arginine for cancer cell survival, and thus can be targeted using arginine-degrading enzymes such as pegylated arginine deiminase (ADI-PEG 20). An initial study by our group of 19 patients demonstrated that a high percentage of SCNEC lack ASS1 expression. Herein, we evaluated an expanded cohort of 74 radical cystectomy patients with HGNEC, including 63 SCNEC, 5 LCNEC, and 6 mixed morphology HGNEC patients. ASS1 expression was assessed through immunohistochemistry. Fifty-eight (of 74, 78%) patients with HGNEC showed absent ASS1 expression, including all patients with LCNEC and mixed morphology (11 of 11, 100%). Ten-year survival from disease-specific death was not statistically significant between ASS1-expressing and ASS1-deficient cases (p = 0.75). Our results show that HGNEC of the bladder may be candidates for arginine deprivation therapy using drugs such as ADI-PEG 20. Further studies are needed to validate these findings and to determine the therapeutic efficacy of such agents.

Acetylglutamate kinase is required for both gametophyte function and embryo development in Arabidopsis thaliana.

The specific functions of the genes encoding arginine biosynthesis enzymes in plants are not well characterized. We report the isolation and characterization of Arabidopsis thaliana N-acetylglutamate kinase (NAGK), which catalyzes the second step of arginine biosynthesis. NAGK is a plastid-localized protein and is expressed during most developmental processes in Arabidopsis. Heterologous expression of the Arabidopsis NAGK gene in a NAGK-deficient Escherichia coli strain fully restores bacterial growth on arginine-deficient medium. nagk mutant pollen tubes grow more slowly than wild type pollen tubes and the phenotype is restored by either specifically through complementation by NAGK in pollen, or exogenous supplementation of arginine. nagk female gametophytes are defective in micropylar pollen tube guidance due to the fact that female gametophyte cell fate specification was specifically affected. Expression of NAGK in synergid cells rescues the defect of nagk female gametophytes. Loss-of-function of NAGK results in Arabidopsis embryos not developing beyond the four-celled embryo stage. The embryo-defective phenotype in nagk/NAGK plants cannot be rescued by watering nagk/NAGK plants with arginine or ornithine supplementation. In conclusion, our results reveal a novel role of NAGK and arginine in regulating gametophyte function and embryo development, and provide valuable insights into arginine transport during embryo development.

Knockdown of a putative argininosuccinate lyase gene reduces arginine content and impairs nymphal development in Nilaparvata lugens.

Nilaparvata lugens is a typical phloem feeder. Rice phloem is high in simple sugars and very low in essential amino acids. Nilaparvata lugens harbors an ascomycete Entomomyces delphacidicola that hypothetically biosynthesizes several amino acids to meet the nutrition requirement of the planthopper. Among these amino acids, here, we focused on arginine biosynthesis. A complete cDNA of an E. delphacidicola gene, arginine-succinate lyase, EdArg4, the last step in arginine biosynthesis, was obtained. RNAi-mediated suppression of EdArg4 reduced arginine content in the hemolymph, and decreased the expression of several arginine biosynthesis genes. Silencing of EdArg4 delayed nymphal development and led to nymphal lethality. About 20% of the EdArg4 RNAi surviving adults were deformed. The most obvious defect was wider and larger abdomen. The EdArg4 RNAi-treated planthoppers had thickened wings and enlarged antennae, legs, and anal tubes and a few adults did not normally emerge. Arginine deficiency in the EdArg4 RNAi planthoppers repressed nitric oxide signaling, determined at the transcriptional level. We infer that E. delphacidicola biosynthesizes essential arginine to compensate for nutrition deficiency in N. lugens.

Genetic and biochemical characterization of arginine biosynthesis in Sinorhizobium meliloti 1021.

L-Ornithine production in the alfalfa microsymbiont Sinorhizobium meliloti occurs as an intermediate step in arginine biosynthesis. Ornithine is required for effective symbiosis but its synthesis in S. meliloti has been little studied. Unlike most bacteria, S. meliloti 1021 is annotated as encoding two enzymes producing ornithine: N-acetylornithine (NAO) deacetylase (ArgE) hydrolyses NAO to acetate and ornithine, and glutamate N-acetyltransferase (ArgJ) transacetylates l-glutamate with the acetyl group from NAO, forming ornithine and N-acetylglutamate (NAG). NAG is the substrate for the second step of arginine biosynthesis catalysed by NAG kinase (ArgB). Inactivation of argB in strain 1021 resulted in arginine auxotrophy. The activity of purified ArgB was significantly inhibited by arginine but not by ornithine. The purified ArgJ was highly active in NAO deacetylation/glutamate transacetylation and was significantly inhibited by ornithine but not by arginine. The purified ArgE protein (with a 6His-Sumo affinity tag) was also active in deacetylating NAO. argE and argJ single mutants, and an argEJ double mutant, are arginine prototrophs. Extracts of the double mutant contained aminoacylase (Ama) activity that deacetylated NAO to form ornithine. The purified products of three candidate ama genes (smc00682 (hipO1), smc02256 (hipO2) and smb21279) all possessed NAO deacetylase activity. hipO1 and hipO2, but not smb21279, expressed in trans functionally complemented an Escherichia coli ΔargE : : Km mutant. We conclude that Ama activity accounts for the arginine prototrophy of the argEJ mutant. Transcriptional assays of argB, argE and argJ, fused to a promoterless gusA gene, showed that their expression was not significantly affected by exogenous arginine or ornithine.

Physiological implications of arginine metabolism in plants.

Nitrogen is a limiting resource for plant growth in most terrestrial habitats since large amounts of nitrogen are needed to synthesize nucleic acids and proteins. Among the 21 proteinogenic amino acids, arginine has the highest nitrogen to carbon ratio, which makes it especially suitable as a storage form of organic nitrogen. Synthesis in chloroplasts via ornithine is apparently the only operational pathway to provide arginine in plants, and the rate of arginine synthesis is tightly regulated by various feedback mechanisms in accordance with the overall nutritional status. While several steps of arginine biosynthesis still remain poorly characterized in plants, much wider attention has been paid to inter- and intracellular arginine transport as well as arginine-derived metabolites. A role of arginine as alternative source besides glutamate for proline biosynthesis is still discussed controversially and may be prevented by differential subcellular localization of enzymes. Apparently, arginine is a precursor for nitric oxide (NO), although the molecular mechanism of NO production from arginine remains unclear in higher plants. In contrast, conversion of arginine to polyamines is well documented, and in several plant species also ornithine can serve as a precursor for polyamines. Both NO and polyamines play crucial roles in regulating developmental processes as well as responses to biotic and abiotic stress. It is thus conceivable that arginine catabolism serves on the one hand to mobilize nitrogen storages, while on the other hand it may be used to fine-tune development and defense mechanisms against stress. This review summarizes the recent advances in our knowledge about arginine metabolism, with a special focus on the model plant Arabidopsis thaliana, and pinpoints still unresolved critical questions.

MoARG1, MoARG5,6 and MoARG7 involved in arginine biosynthesis are essential for growth, conidiogenesis, sexual reproduction, and pathogenicity in Magnaporthe oryzae

Arginine is one of the most versatile amino acids in eukaryote cells, which plays important roles in a multitude of processes such as protein synthesis, nitrogen metabolism, nitric oxide (NO) and urea biosynthesis. The de novo arginine biosynthesis pathway is conserved among fungal kingdom, but poorly understood in plant pathogenic fungi. Here, we characterized the functions of three synthetic enzyme-encoding genes MoARG1, MoARG5,6, and MoARG7, which involved the seventh step, second–third step and fifth step of arginine biosynthesis in Magnaporthe oryzae, respectively. Deletion of MoARG1 or MoARG5,6, resulted in arginine auxotrophic mutants, which had a strict requirement for arginine on minimal medium (MM). Both ΔMoarg1 and ΔMoarg5,6 severely reduced in aerial hyphal growth, pigmentation, conidiogenesis, sexual reproduction and pathogenicity. Interestingly, like Saccharomyces cerevisiae, deletion of MoARG7 caused a leaky arginine auxotrophy, and attenuated pathogenicity. Limited appressorium-mediated penetration and restricted invasive hyphae growth in host cells are responsible for the severely attenuated pathogenicity of the Arg− mutants. Additionally, we monitored the NO generation during conidial germination and appressorial formation in both Arg− mutants and wild type, and demonstrated that NO generation may not occur via arginine-dependent pathway in M. oryzae. In summary, MoARG1, MoARG5,6, and MoARG7 are required for growth, conidiogenesis, sexual reproduction, and pathogenicity in M. oryzae.

Argininosuccinate synthetase 1 (ASS1) expression and response to arginine deprivation with ADI-PEG20 in human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC).

e17041 Background: Proteomic studies determined that ASS1, the metabolic enzyme which catalyzes the penultimate step of arginine biosynthesis, is differentially expressed in HNSCC tumors with respe...

Normal root elongation requires arginine produced by argininosuccinate lyase in rice

Plant roots play an important role in the uptake of water and nutrients, structural support and environmental sensing, but the molecular mechanisms involved in root development are poorly understood in rice (Oryza sativa), which is characterized by a dense fibrous root system. Here we report a rice mutant (red1 for root elongation defect 1) with short roots. Morphological and physiological analyses showed that the mutant had a shorter length from the quiescent center (QC) to the starting point of the elongation zone but a similar cell size and number of lateral and crown roots compared with the wild type. Furthermore, the mutant had similar radial structure and nutrient uptake patterns to the wild type. Map-based cloning revealed that the mutant phenotype was caused by a point mutation of a gene encoding an argininosuccinate lyase (ASL), catalyzing the last step of arginine biosynthesis. The OsASL1 gene has two distinct transcripts, OsASL1.1 and OsASL1.2, which result from different transcription start sites, but only OsASL1.1 was able to complement the mutant phenotype. OsASL1.1 was expressed in both the roots and shoots. The protein encoded by OsASL1.1 showed ASL activity in yeast. OsALS1.1 was localized to the plastid. The short root of the mutant was rescued by exogenous addition of arginine, but not by other amino acids. These results indicate that arginine produced by ASL is required for normal root elongation in rice.

A novel type of N-acetylglutamate synthase is involved in the first step of arginine biosynthesis in Corynebacterium glutamicum

BackgroundArginine biosynthesis in Corynebacterium glutamicum consists of eight enzymatic steps, starting with acetylation of glutamate, catalysed by N-acetylglutamate synthase (NAGS). There are different kinds of known NAGSs, for example, “classical” ArgA, bifunctional ArgJ, ArgO, and S-NAGS. However, since C. glutamicum possesses a monofunctional ArgJ, which catalyses only the fifth step of the arginine biosynthesis pathway, glutamate must be acetylated by an as of yet unknown NAGS gene.ResultsArginine biosynthesis was investigated by metabolome profiling using defined gene deletion mutants that were expected to accumulate corresponding intracellular metabolites. HPLC-ESI-qTOF analyses gave detailed insights into arginine metabolism by detecting six out of seven intermediates of arginine biosynthesis. Accumulation of N-acetylglutamate in all mutants was a further confirmation of the unknown NAGS activity. To elucidate the identity of this gene, a genomic library of C. glutamicum was created and used to complement an Escherichia coli ΔargA mutant. The plasmid identified, which allowed functional complementation, contained part of gene cg3035, which contains an acetyltransferase domain in its amino acid sequence. Deletion of cg3035 in the C. glutamicum genome led to a partial auxotrophy for arginine. Heterologous overexpression of the entire cg3035 gene verified its ability to complement the E. coli ΔargA mutant in vivo and homologous overexpression led to a significantly higher intracellular N-acetylglutamate pool. Enzyme assays confirmed the N-acetylglutamate synthase activity of Cg3035 in vitro. However, the amino acid sequence of Cg3035 revealed no similarities to members of known NAGS gene families.ConclusionsThe N-acetylglutamate synthase Cg3035 is able to catalyse the first step of arginine biosynthesis in C. glutamicum. It represents a novel class of NAGS genes apparently present only in bacteria of the suborder Corynebacterineae, comprising amongst others the genera Corynebacterium, Mycobacterium, and Nocardia. Therefore, the name C-NAGS (Corynebacterineae-type NAGS) is proposed for this new family.

Epigenetic inactivation to target the arginine biosynthetic pathway in multiple myeloma.

e18567 Background: Argininoosuccinate synthetase-1 (ASS1) catalyses the rate-limiting step in arginine biosynthesis, the conversion of citruline to arginine. Argininosuccinate lyase (ASL) is an enzyme that catalyses the reversible breakdown of arginosuccinate producing arginine and fumerate. Arginine deprivation using arginine deiminase (ADI-PEG20) is currently considered as a novel therapeutic intervention for cancer. In this perspective, we investigated the methylation status of the ASS1 and ASL CpG islands in multiple myeloma (MM) and analyzed for clinical relevance. Methods: Genomic DNA was extracted from bone marrow aspirate samples from 46 MM patients (28 male, 18 female, median age 64 years) obtained at diagnosis. Methylation-specific PCR was employed to study the methylation in the ASS1 and ASL CpG islands. DNA was isolated and bisulphite modified using commercially available kits. Logistic regression analyses were used to measure the association between gene methylation and sex, age>65, ISS stage, presence of extramedullary disease, renal failure and bone disease. Kaplan-Meier curves were used to estimate the probabilities of survival and the Log-rank test to assess the statistical significance of differences in event rates. Results: Methylation in the CpG island of ASS1 was detected in 71.7% patients and of ASL in 37% patients, while simultaneous methylation in both genes was present in 10 patients. None of the two genes was detectably methylated in the control group. Patients with ASS1 methylation were less likely to have bone disease (p=0.04, OR=0.22) and extramedullary disease (p=0.05, OR=0.22) and a trend was also noted that these patients were less likely to be >65 years of age (p=0.2, OR=0.37). We did not detect any statistically significant difference in overall survival by methylation status of the studied genes in this small study size. Conclusions: We demonstrate for the first time that arginine biosynthesis genes ASS1 and ASL are methylated in MM.Methylation of ASS1 was found to be more frequent and negatively associated with bone or extramedullary disease. Further evaluation of ASS1 and ASL is warranted in MM and supports the expansion of arginine deprivation trials in these patients

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.

Structural insights into regulation of vertebrate homolog N‐acetylglutamate synthase/kinase 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. The crystal structures of M. maris NAGS/K (mmNAGS/K) with and without L‐arginine bound indicate that mmNAGS/K functions as a tetramer, in contrast to the hexameric structure of Neisseria gonorrhoeae NAGS. Each subunit has an amino acid kinase (AAK) domain, which is likely responsible for N‐acetylglutamate kinase (NAGK) activity and has a arginine binding site, and an N‐acetyltransferase (NAT) domain that contains the putative NAGS active site. 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.

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.

Epigenetic Inactivation Targets the Arginine Biosynthetic Pathway At Two Levels in Multiple Myeloma

Abstract 4640 Background.Arginine is critical for the growth of certain human cancers and is an intermediary in the synthesis of various bioactive molecules. Argininosuccinate synthetase-1 (ASS1) catalyses the rate-limiting step in arginine biosynthesis, the conversion of citruline to arginine. Argininosuccinate lyase (ASL) is an enzyme that catalyses the reversible breakdown of arginosuccinate producing arginine and fumerate. Down-regulation of ASS1 has been demonstrated in several solid cancers and has been shown to confer sensitivity to arginine deprivation (arginine auxotrophic tumors). Methylation-dependent silencing of the ASS1 promoter may account for ASS1 down-regulation and arginine auxotrophy. Arginine deprivation using arginine deiminase (ADI-PEG20) is currently considered as a novel therapeutic intervention for cancer, and early clinical trials have shown promising results in solid tumors. In this perspective, we investigated for the first time the methylation status of the ASS1 and ASL CpG islands in multiple myeloma (MM) and analyzed for clinical relevance. Methods.Genomic DNA was extracted from bone marrow aspirate samples from 46 MM patients (28 male, 18 female, median age 64 years) obtained at diagnosis. Methylation-specific PCR (MSP) was employed to study the methylation in the ASS1 and ASL CpG islands. DNA was isolated and bisulphite modified using commercially available kits (QIAmp DNA mini kit, Qiagen and EZ DNA methylation kit, Zymo Research respectively). Control methylated and unmethylated genomic DNAs were included in each experiment. Ten bone marrow samples, with no neoplastic cells, from patients with borderline thrombocytopenia served as negative controls. Logistic regression analysis was used to measure the association between gene methylation and sex, age, ISS stage, presence of extramedullary disease, renal failure (eGFR<50 ml/min) and bone disease. Results.Methylation in the CpG island of ASS1 was detected in 71.7% patients (95% CI 57–82%) and of ASL in 37% patients (95% CI 24–51%), while simultaneous methylation in both genes was present in 10 patients. None of the two genes were detectably methylated in the control group. Patients with ASS1 methylation were less likely to have bone disease (p=0.04, OR=0.22) and extramedullary disease (p=0.05, OR=0.22). There was no statistically significant difference in overall survival by methylation status of the studied genes. Conclusions.Arginine biosynthesis genes ASS1 and ASL are methylated in MM. Methylation of ASS1 was found to be more frequent and negatively associated with bone or extramedullary disease. Further evaluation of ASS1 and ASL methylation is warranted in MM in the perspective of expanding arginine deprivation trials in these patients Disclosures:No relevant conflicts of interest to declare.

Two Crystal Structures of Escherichia coli N-Acetyl-l-Glutamate Kinase Demonstrate the Cycling between Open and Closed Conformations

N-Acetyl-l-glutamate kinase (NAGK), the paradigm enzyme of the amino acid kinase family, catalyzes the second step of arginine biosynthesis. Although substrate binding and catalysis were clarified by the determination of four crystal structures of the homodimeric Escherichia coli enzyme (EcNAGK), we now determine 2 Å resolution crystal structures of EcNAGK free from substrates or complexed with the product N-acetyl-l-glutamyl-5-phosphate (NAGP) and with sulfate, which reveal a novel, very open NAGK conformation to which substrates would associate and from which products would dissociate. In this conformation, the C-domain, which hosts most of the nucleotide site, rotates ∼24°–28° away from the N-domain, which hosts the acetylglutamate site, whereas the empty ATP site also exhibits some changes. One sulfate is found binding in the region where the β-phosphate of ATP normally binds, suggesting that ATP is first anchored to the β-phosphate site, before perfect binding by induced fit, triggering the shift to the closed conformation. In contrast, the acetylglutamate site is always well formed, although its β-hairpin lid is found here to be mobile, being closed only in the subunit of the EcNAGK–NAGP complex that binds NAGP most strongly. Lid closure appears to increase the affinity for acetylglutamate/NAGP and to stabilize the closed enzyme conformation via lid–C-domain contacts. Our finding of NAGP bound to the open conformation confirms that this product dissociates from the open enzyme form and allows reconstruction of the active center in the ternary complex with both products, delineating the final steps of the reaction, which is shown here by site-directed mutagenesis to involve centrally the invariant residue Gly11.

Characterization of a gene encoding ornithine carbamoyltransferase from rice

Ornithinine carbamoyltransferase (OTC) is an enzyme that catalyzes the key step in arginine biosynthesis in bacteria and plants. OTC is also involved in the urea cycle and deficiency of the enzyme in human leads to disease. The argF gene encoding OTC has been reported in many bacteria and few plants. Here we report the characterization of a gene encoding OTC from rice (OsOTC). Analysis of a cDNA sequence from rice revealed that the full-length open reading frame of OsOTC consisted of 367 amino acids, corresponding to a protein of approximately 39.7 kDa. The predicted amino acid sequence of OsOTC harbor distinct five OTC signature sites and is highly homologous to that of enzymes of plants, animals and many bacterial OTCs. Expression of OsOTC in argF mutants of Escherichia coli showed that the gene was able to functionally complement to the mutant. These results suggest that the OsOTC encode a protein for ornithine carbamoyltransferase in rice.

N-Acetyl-l-Glutamate Kinase (NAGK) from Oxygenic Phototrophs: PII Signal Transduction across Domains of Life Reveals Novel Insights in NAGK Control

N-Acetyl-l-glutamate kinase (NAGK) catalyzes the first committed step in arginine biosynthesis in organisms that perform the cyclic pathway of ornithine synthesis. In eukaryotic and bacterial oxygenic phototrophs, the activity of NAGK is controlled by the PII signal transduction protein. Recent X-ray analysis of NAGK–PII complexes from a higher plant (Arabidopsis thaliana) and a cyanobacterium (Synechococcus elongatus) revealed that despite several differences, the overall structure of the complex is highly similar. The present study analyzes the functional conservation of PII-mediated NAGK regulation in plants and cyanobacteria to distinguish between universal properties and those that are specific for the different phylogenetic lineages. This study shows that plant and cyanobacterial PII proteins can mutually regulate the NAGK enzymes across the domains of life, implying a high selective pressure to conserve PII–NAGK interaction over more than 1.2 billion years of separate evolution. The non-conserved C-terminus of S. elongatus NAGK was identified as an element, which strongly enhances arginine inhibition and is responsible for most of the differences between S. elongatus and A. thaliana NAGK with respect to arginine sensitivity. Both PII proteins relieve arginine inhibition of NAGK, and in both lineages, PII-mediated relief from arginine inhibition is antagonized by 2-oxoglutarate. Together, these properties highlight the conserved role of PII as a signal integrator of the C/N balance sensed as 2-oxoglutarate to regulate arginine synthesis in oxygenic phototrophs.

Basis of Arginine Sensitivity of Microbial N -Acetyl- l -Glutamate Kinases: Mutagenesis and Protein Engineering Study with the Pseudomonas aeruginosa and Escherichia coli Enzymes

N-acetylglutamate kinase (NAGK) catalyzes the second step of arginine biosynthesis. In Pseudomonas aeruginosa, but not in Escherichia coli, this step is rate limiting and feedback and sigmoidally inhibited by arginine. Crystal structures revealed that arginine-insensitive E. coli NAGK (EcNAGK) is homodimeric, whereas arginine-inhibitable NAGKs, including P. aeruginosa NAGK (PaNAGK), are hexamers in which an extra N-terminal kinked helix (N-helix) interlinks three dimers. By introducing single amino acid replacements in PaNAGK, we prove the functionality of the structurally identified arginine site, as arginine site mutations selectively decreased the apparent affinity for arginine. N-helix mutations affecting R24 and E17 increased and decreased, respectively, the apparent affinity of PaNAGK for arginine, as predicted from enzyme structures that revealed the respective formation by these residues of bonds favoring inaccessible and accessible arginine site conformations. N-helix N-terminal deletions spanning > or = 16 residues dissociated PaNAGK to active dimers, those of < or = 20 residues decreased the apparent affinity for arginine, and complete N-helix deletion (26 residues) abolished arginine inhibition. Upon attachment of the PaNAGK N-terminal extension to the EcNAGK N terminus, EcNAGK remained dimeric and arginine insensitive. We concluded that the N-helix and its C-terminal portion after the kink are essential but not sufficient for hexamer formation and arginine inhibition, respectively; that the N-helix modulates NAGK affinity for arginine and mediates signal transmission between arginine sites, thus establishing sigmoidal arginine inhibition kinetics; that the mobile alphaH-beta16 loop of the arginine site is the modulatory signal receiver; and that the hexameric architecture is not essential for arginine inhibition but is functionally essential for physiologically relevant arginine control of NAGK.

Structural Basis for the Regulation of N-Acetylglutamate Kinase by PII in Arabidopsis thaliana

PII is a highly conserved regulatory protein found in organisms across the three domains of life. In cyanobacteria and plants, PII relieves the feedback inhibition of the rate-limiting step in arginine biosynthesis catalyzed by N-acetylglutamate kinase (NAGK). To understand the molecular structural basis of enzyme regulation by PII, we have determined a 2.5-A resolution crystal structure of a complex formed between two homotrimers of PII and a single hexamer of NAGK from Arabidopsis thaliana bound to the metabolites N-acetylglutamate, ADP, ATP, and arginine. In PII, the T-loop and Trp(22) at the start of the alpha1-helix, which are both adjacent to the ATP-binding site of PII, contact two beta-strands as well as the ends of two central helices (alphaE and alphaG) in NAGK, the opposing ends of which form major portions of the ATP and N-acetylglutamate substrate-binding sites. The binding of Mg(2+).ATP to PII stabilizes a conformation of the T-loop that favors interactions with both open and closed conformations of NAGK. Interactions between PII and NAGK appear to limit the degree of opening and closing of the active-site cleft in opposition to a domain-separating inhibitory effect exerted by arginine, thus explaining the stimulatory effect of PII on the kinetics of arginine-inhibited NAGK.