RESUMEN
Cancer is a leading concern and important cause of death worldwide. Cancer is a non-communicable illness defined as uncontrolled division of cells. It can develop into metastatic cancer when tumor cells migrate to other organs. In recent years evidence has emerged that the bioavailability of Asn play a crucial role in cancer metastasis. Asn is a non-essential amino acid formed from an ATP dependent catalyzed reaction by the enzyme asparagine synthetase (ASNS), where Asp and Gln are converted to Asn and Glu, respectively. The human ASNS enzyme consist of 561 amino acids, with a molecular weight of 64 KDa. ASNS governs the activation of transcriptional factors that regulate the process of metastasis. In this work the 3D model of ASNS in E. coli (AS-B) and the human ASNS docked with its different ligands have been used to study the 3D mechanism of the conversion of Asp and Gln to Asn and Glu, in human ASNS. The stability evaluation of the docked complexes was checked by molecular dynamic simulation through the bioinformatic tool Desmond. The binding residues and their interactions can be exploited for the development of inhibitors, as well as for finding new drug molecules against ASNS and prevention of metastatic cancer.
Asunto(s)
Aspartatoamoníaco Ligasa , Dominio Catalítico , Simulación de Dinámica Molecular , Humanos , Aspartatoamoníaco Ligasa/metabolismo , Aspartatoamoníaco Ligasa/química , Aspartatoamoníaco Ligasa/genética , Simulación del Acoplamiento Molecular , Especificidad por Sustrato , Asparagina/metabolismo , Asparagina/química , Unión Proteica , Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/enzimología , Simulación por Computador , Ligandos , Ácido Aspártico/metabolismo , Ácido Aspártico/química , Ligasas de Carbono-Nitrógeno con Glutamina como Donante de Amida-NRESUMEN
ß-Aspartyl compounds, such as ß-aspartyl hydroxamate (serine racemase inhibitor), ß-aspartyl-l-lysine (moisture retention), and ß-aspartyl-l-tryptophan (immunomodulator) are physiologically active compounds. There is limited literature on the development of effective methods of production of ß-aspartyl compounds. In this study, we describe the biochemical characterization of asparagine synthetase (AS) from Streptococcus thermophilus NBRC 13957 (StAS) and the enzymatic synthesis of ß-aspartyl compounds using StAS. Recombinant StAS was expressed in Escherichia coli BL21(DE3) and it displayed activity towards hydroxylamine, methylamine, ethylamine, and ammonia, as acceptors of the ß-aspartyl moiety. StAS exhibited higher activity toward hydroxylamine and ethylamine as acceptor substrates compared with the enzymes from Lactobacillus delbrueckii NBRC 13953, Lactobacillus reuteri NBRC 15892, and E. coli. The coupling of the synthesis of ß-aspartyl compounds by StAS with an ATP-regeneration system using polyphosphate kinase from Deinococcus proteoliticus NBRC 101906 displayed an approximately 2.5-fold increase in the production of ß-aspartylhydroxamate from 1.06 mM to 2.53 mM after a 76-h reaction.
Asunto(s)
Aspartatoamoníaco Ligasa , Escherichia coli , Proteínas Recombinantes , Streptococcus thermophilus , Streptococcus thermophilus/enzimología , Streptococcus thermophilus/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Aspartatoamoníaco Ligasa/metabolismo , Aspartatoamoníaco Ligasa/genética , Aspartatoamoníaco Ligasa/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Ácido Aspártico/metabolismo , Ácido Aspártico/biosíntesis , Especificidad por Sustrato , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/químicaRESUMEN
The natural amino acid asparagine (Asn) is required by cells to sustain function and proliferation. Healthy cells can synthesize Asn through asparagine synthetase (ASNS) activity, whereas specific cancer and genetically diseased cells are forced to obtain asparagine from the extracellular environment. ASNS catalyzes the ATP-dependent synthesis of Asn from aspartate by consuming glutamine as a nitrogen source. Asparagine Synthetase Deficiency (ASNSD) is a disease that results from biallelic mutations in the ASNS gene and presents with congenital microcephaly, intractable seizures, and progressive brain atrophy. ASNSD often leads to premature death. Although clinical and cellular studies have reported that Asn deprivation contributes to the disease symptoms, the global metabolic effects of Asn deprivation on ASNSD-derived cells have not been studied. We analyzed two previously characterized cell culture models, lymphoblastoids and fibroblasts, each carrying unique ASNS mutations from families with ASNSD. Metabolomics analysis demonstrated that Asn deprivation in ASNS-deficient cells led to disruptions across a wide range of metabolites. Moreover, we observed significant decrements in TCA cycle intermediates and anaplerotic substrates in ASNS-deficient cells challenged with Asn deprivation. We have identified pantothenate, phenylalanine, and aspartate as possible biomarkers of Asn deprivation in normal and ASNSD-derived cells. This work implies the possibility of a novel ASNSD diagnostic via targeted biomarker analysis of a blood draw.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos , Aspartatoamoníaco Ligasa , Discapacidad Intelectual , Microcefalia , Humanos , Asparagina/genética , Aspartatoamoníaco Ligasa/genética , Aspartatoamoníaco Ligasa/química , Aspartatoamoníaco Ligasa/metabolismo , Ácido Aspártico , Discapacidad Intelectual/genética , AtrofiaRESUMEN
Asparagine synthetase (ASNS) catalyzes synthesis of asparagine (Asn) and Glu from Asp and Gln in an ATP-dependent reaction. Asparagine synthetase deficiency (ASNSD) results from biallelic mutations in the ASNS gene. Affected children exhibit congenital microcephaly, continued brain atrophy, seizures, and often premature mortality. However, the underlying mechanisms are unclear. This report describes a compound heterozygotic ASNSD child with two novel mutations in the ASNS gene, c.1118G>T (paternal) and c.1556G>A (maternal), that lead to G373V or R519H ASNS variants. Structural mapping suggested that neither variant participates directly in catalysis. Growth of cultured fibroblasts from either parent was unaffected in Asn-free medium, whereas growth of the child's cells was suppressed by about 50%. Analysis of Asn levels unexpectedly revealed that extracellular rather than intracellular Asn correlated with the reduced proliferation during incubation of the child's cells in Asn-free medium. Our attempts to ectopically express the G373V variant in either HEK293T or JRS cells resulted in minimal protein production, suggesting instability. Protein expression and purification from HEK293T cells revealed reduced activity for the R519H variant relative to WT ASNS. Expression of WT ASNS in ASNS-null JRS cells resulted in nearly complete rescue of growth in Asn-free medium, whereas we observed no proliferation for the cells expressing either the G373V or R519H variant. These results support the conclusion that the coexpression of the G373V and R519H ASNS variants leads to significantly reduced Asn synthesis, which negatively impacts cellular growth. These observations are consistent with the ASNSD phenotype.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos , Aspartatoamoníaco Ligasa , Discapacidad Intelectual , Microcefalia , Enfermedades Neurodegenerativas , Adenosina Trifosfato , Asparagina/genética , Aspartatoamoníaco Ligasa/química , Atrofia , Ligasas de Carbono-Nitrógeno con Glutamina como Donante de Amida-N/genética , Niño , Células HEK293 , Humanos , Discapacidad Intelectual/genética , Microcefalia/genética , MutaciónRESUMEN
Asparagine synthetase (AS), a key enzyme in plant nitrogen metabolism, plays an important role in plant nitrogen assimilation and distribution. Asparagine (Asn), the product of asparagine synthetase, is one of the main compounds responsible for organic nitrogen transport and storage in plants. In this study, we performed complementation experiments using an Asn-deficient Escherichia coli strain to demonstrate that three putative asparagine synthetase family members in poplar (Populussimonii× P.nigra) function in Asn synthesis. Quantitative real-time PCR revealed that the three members had high expression levels in different tissues of poplar and were regulated by exogenous nitrogen. PnAS1 and PnAS2 were also affected by diurnal rhythm. Long-term dark treatment resulted in a significant increase in PnAS1 and PnAS3 expression levels. Under long-term light conditions, however, PnAS2 expression decreased significantly in the intermediate region of leaves. Exogenous application of ammonium nitrogen, glutamine, and a glutamine synthetase inhibitor revealed that PnAS3 was more sensitive to exogenous glutamine, while PnAS1 and PnAS2 were more susceptible to exogenous ammonium nitrogen. Our results suggest that the various members of the PnAS gene family have distinct roles in different tissues and are regulated in different ways.
Asunto(s)
Asparagina/metabolismo , Aspartatoamoníaco Ligasa/genética , Nitrógeno/metabolismo , Populus/enzimología , Aspartatoamoníaco Ligasa/química , Escherichia coli/genética , Regulación de la Expresión Génica de las Plantas , Glutamato-Amoníaco Ligasa/química , Glutamato-Amoníaco Ligasa/genética , Glutamina/metabolismo , Populus/genéticaRESUMEN
l-Asparagine synthetase (AS) acts in asparagine formation and can be classified into two families: AS-A or AS-B. AS-A is mainly found in prokaryotes and can synthetize asparagine from ammonia. Distinct from other eukaryotes, Trypanosoma cruzi produces an AS-A. AS-A from Trypanosoma cruzi (Tc-AS-A) differs from prokaryotic AS-A due to its ability to catalyze asparagine synthesis using both glutamine and ammonia as nitrogen sources. Regarding these peculiarities, this work uses several biophysical techniques to provide data concerning the Tc-AS-A in-solution behavior. Tc-AS-A was produced as a recombinant and purified by three chromatography steps. Circular dichroism, dynamic light scattering, and analytical size exclusion chromatography showed that Tc-AS-A has the same fold and quaternary arrangement of prokaryotic AS-A. Despite the tendency of protein to aggregate, stable dimers were obtained when solubilization occurred at pH ≤ 7.0. We also demonstrate the protective efficacy against T. cruzi infection in mice immunized with Tc-AS-A. Our results indicate that immunization with Tc-AS-A might confer partial protection to infective forms of T. cruzi in this particular model.
Asunto(s)
Asparagina/metabolismo , Aspartatoamoníaco Ligasa/inmunología , Aspartatoamoníaco Ligasa/metabolismo , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Trypanosoma cruzi/enzimología , Amoníaco/metabolismo , Animales , Aspartatoamoníaco Ligasa/química , Aspartatoamoníaco Ligasa/aislamiento & purificación , Enfermedad de Chagas/prevención & control , Cromatografía Liquida , Dicroismo Circular , Modelos Animales de Enfermedad , Dispersión Dinámica de Luz , Glutamina/metabolismo , Ratones Endogámicos BALB C , Parasitemia/prevención & control , Conformación Proteica , Pliegue de Proteína , Vacunas Antiprotozoos/administración & dosificación , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Vacunas Sintéticas/administración & dosificaciónRESUMEN
Recent work has found that many metabolic enzymes have the ability to polymerize in response to metabolic changes or environmental stress. This ability to polymerize is well conserved for the few metabolic enzyme paralogs that have been studied in yeast. Here we describe the first set of paralogs, Asn1p and Asn2p, that have differential assembly behavior. Asn1p and Asn2p both co-assemble into filaments in response to nutrient limitation. However, the ability of Asn2p to form filaments is strictly dependent on the presence of Asn1p. Using mutations that block enzyme activity but have differential effects on Asn1p polymerization, we have found that Asn1p polymers are unlikely to have acquired a moonlighting function. Together these results provide a novel system for understanding the regulation and evolution of metabolic enzyme polymerization.
Asunto(s)
Aspartatoamoníaco Ligasa/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Estrés Fisiológico , Aspartatoamoníaco Ligasa/química , Aspartatoamoníaco Ligasa/genética , Mutación , Nutrientes , Polimerizacion , Conformación Proteica , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Asparagine synthetase (ASNS) converts aspartate and glutamine to asparagine and glutamate in an ATP-dependent reaction. ASNS is present in most, if not all, mammalian organs, but varies widely in basal expression. Human ASNS activity is highly responsive to cellular stress, primarily by increased transcription from a single gene located on chromosome 7. Elevated ASNS protein expression is associated with resistance to asparaginase therapy in childhood acute lymphoblastic leukemia. There is evidence that ASNS expression levels may also be inversely correlated with asparaginase efficacy in certain solid tumors as well. Children with mutations in the ASNS gene exhibit developmental delays, intellectual disability, microcephaly, intractable seizures, and progressive brain atrophy. Thus far, 15 unique mutations in the ASNS gene have been clinically associated with asparagine synthetase deficiency (ASD). Molecular modeling using the Escherichia coli ASNS-B structure has revealed that most of the reported ASD substitutions are located near catalytic sites or within highly conserved regions of the protein. For some ASD patients, fibroblast cell culture studies have eliminated protein and mRNA synthesis or stability as the basis for decreased proliferation.
Asunto(s)
Aspartatoamoníaco Ligasa/genética , Regulación Enzimológica de la Expresión Génica , Mutación , Animales , Asparaginasa/uso terapéutico , Aspartatoamoníaco Ligasa/química , Aspartatoamoníaco Ligasa/fisiología , Resistencia a Medicamentos/genética , Predisposición Genética a la Enfermedad , HumanosRESUMEN
A growing interest in asparagine (Asn) metabolism has currently been observed in cancer and infection fields. Asparagine synthetase (AS) is responsible for the conversion of aspartate into Asn in an ATP-dependent manner, using ammonia or glutamine as a nitrogen source. There are two structurally distinct AS: the strictly ammonia dependent, type A, and the type B, which preferably uses glutamine. Absent in humans and present in trypanosomatids, AS-A was worthy of exploring as a potential drug target candidate. Appealingly, it was reported that AS-A was essential in Leishmania donovani, making it a promising drug target. In the work herein we demonstrate that Leishmania infantum AS-A, similarly to Trypanosoma spp. and L. donovani, is able to use both ammonia and glutamine as nitrogen donors. Moreover, we have successfully generated LiASA null mutants by targeted gene replacement in L. infantum, and these parasites do not display any significant growth or infectivity defect. Indeed, a severe impairment of in vitro growth was only observed when null mutants were cultured in asparagine limiting conditions. Altogether our results demonstrate that despite being important under asparagine limitation, LiAS-A is not essential for parasite survival, growth or infectivity in normal in vitro and in vivo conditions. Therefore we exclude AS-A as a suitable drug target against L. infantum parasites.
Asunto(s)
Aspartatoamoníaco Ligasa/metabolismo , Leishmania infantum/enzimología , Leishmania infantum/patogenicidad , Leishmaniasis Visceral/parasitología , Proteínas Protozoarias/metabolismo , Amoníaco/metabolismo , Animales , Asparagina/metabolismo , Aspartatoamoníaco Ligasa/química , Aspartatoamoníaco Ligasa/genética , Femenino , Glutamina/metabolismo , Humanos , Leishmania infantum/genética , Leishmania infantum/crecimiento & desarrollo , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Alineación de Secuencia , VirulenciaRESUMEN
Asparagine Synthetase Deficiency is a recently described cause of profound intellectual disability, marked progressive cerebral atrophy and variable seizure disorder. To date there has been limited functional data explaining the underlying pathophysiology. We report a new case with compound heterozygous mutations in the ASNS gene (NM_183356.3:c. [866G>C]; [1010C>T]). Both variants alter evolutionarily conserved amino acids and were predicted to be pathogenic based on in silico protein modelling that suggests disruption of the critical ATP binding site of the ASNS enzyme. In patient fibroblasts, ASNS expression as well as protein and mRNA stability are not affected by these variants. However, there is markedly reduced proliferation of patient fibroblasts when cultured in asparagine-limited growth medium, compared to parental and wild type fibroblasts. Restricting asparagine replicates the physiology within the blood-brain-barrier, with limited transfer of dietary derived asparagine, resulting in reliance of neuronal cells on intracellular asparagine synthesis by the ASNS enzyme. These functional studies offer insight into the underlying pathophysiology of the dramatic progressive cerebral atrophy associated with Asparagine Synthetase Deficiency.
Asunto(s)
Asparagina/metabolismo , Aspartatoamoníaco Ligasa/deficiencia , Aspartatoamoníaco Ligasa/genética , Proliferación Celular , Mutación , Adenosina Trifosfato/metabolismo , Aspartatoamoníaco Ligasa/química , Aspartatoamoníaco Ligasa/metabolismo , Sitios de Unión , Células Cultivadas , Simulación por Computador , Medios de Cultivo/química , Exoma , Femenino , Fibroblastos/patología , Humanos , Discapacidad Intelectual/etiología , Discapacidad Intelectual/genética , Masculino , Análisis de Secuencia de ADNRESUMEN
Asparagine is synthesized from glutamine by the reaction of asparagine synthetase (AS) and is the major nitrogen form in both xylem and phloem sap in rice (Oryza sativa L.). There are two genes encoding AS, OsAS1 and OsAS2, in rice, but the functions of individual AS isoenzymes are largely unknown. Cell type- and NH4(+)-inducible expression of OsAS1 as well as analyses of knockout mutants were carried out in this study to characterize AS1. OsAS1 was mainly expressed in the roots, with in situ hybridization showing that the corresponding mRNA was specifically accumulated in the three cell layers of the root surface (epidermis, exodermis and sclerenchyma) in an NH4(+)-dependent manner. Conversely, OsAS2 mRNA was abundant in leaf blades and sheathes of rice. Although OsAS2 mRNA was detectable in the roots, its content decreased when NH4(+) was supplied. Retrotransposon-mediated knockout mutants lacking AS1 showed slight stimulation of shoot length and slight reduction in root length at the seedling stage. On the other hand, the mutation caused an approximately 80-90% reduction in free asparagine content in both roots and xylem sap. These results suggest that AS1 is responsible for the synthesis of asparagine in rice roots following the supply of NH4(+). Characteristics of the NH4(+)-dependent increase and the root surface cell-specific expression of OsAS1 gene are very similar to our previous results on cytosolic glutamine synthetase1;2 and NADH-glutamate synthase1 in rice roots. Thus, AS1 is apparently coupled with the primary assimilation of NH4(+) in rice roots.
Asunto(s)
Compuestos de Amonio/farmacología , Asparagina/biosíntesis , Aspartatoamoníaco Ligasa/metabolismo , Oryza/enzimología , Proteínas de Plantas/metabolismo , Raíces de Plantas/enzimología , Secuencia de Aminoácidos , Aspartatoamoníaco Ligasa/química , Carbono/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Técnicas de Inactivación de Genes , Genes de Plantas , Meristema/efectos de los fármacos , Meristema/metabolismo , Datos de Secuencia Molecular , Mutación , Nitrógeno/metabolismo , Oryza/efectos de los fármacos , Oryza/genética , Fenotipo , Proteínas de Plantas/química , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/genética , Brotes de la Planta/efectos de los fármacos , Brotes de la Planta/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Plantones/efectos de los fármacos , Plantones/genéticaRESUMEN
Asparagine is formed by two structurally distinct asparagine synthetases in prokaryotes. One is the ammonia-utilizing asparagine synthetase A (AsnA), and the other is asparagine synthetase B (AsnB) that uses glutamine or ammonia as a nitrogen source. In a previous investigation using sequence-based analysis, we had shown that Leishmania spp. possess asparagine-tRNA synthetase paralog asparagine synthetase A (LdASNA) that is ammonia-dependent. Here, we report the cloning, expression, and kinetic analysis of ASNA from Leishmania donovani. Interestingly, LdASNA was both ammonia- and glutamine-dependent. To study the physiological role of ASNA in Leishmania, gene deletion mutations were attempted via targeted gene replacement. Gene deletion of LdASNA showed a growth delay in mutants. However, chromosomal null mutants of LdASNA could not be obtained as the double transfectant mutants showed aneuploidy. These data suggest that LdASNA is essential for survival of the Leishmania parasite. LdASNA enzyme was recalcitrant toward crystallization so we instead crystallized and solved the atomic structure of its close homolog from Trypanosoma brucei (TbASNA) at 2.2 Å. A very significant conservation in active site residues is observed between TbASNA and Escherichia coli AsnA. It is evident that the absence of an LdASNA homolog from humans and its essentiality for the parasites make LdASNA a novel drug target.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Aspartatoamoníaco Ligasa/metabolismo , Bacterias/enzimología , Leishmania donovani/enzimología , Secuencia de Aminoácidos , Aminoacil-ARNt Sintetasas/química , Aminoacil-ARNt Sintetasas/genética , Animales , Aspartatoamoníaco Ligasa/química , Aspartatoamoníaco Ligasa/genética , Secuencia de Bases , Cartilla de ADN , Microscopía Confocal , Modelos Moleculares , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Fracciones Subcelulares/enzimologíaRESUMEN
Lactobacillus casei ATCC 27139 enhances host innate immunity, and the J1 phage-resistant mutants of this strain lose the activity. A transposon insertion mutant library of L. casei ATCC 27139 was constructed, and nine J1 phage-resistant mutants out of them were obtained. Cloning and sequencing analyses identified three independent genes that were disrupted by insertion of the transposon element: asnH, encoding asparagine synthetase, and dnaJ and dnaK, encoding the molecular chaperones DnaJ and DnaK, respectively. Using an in vivo mouse model of Listeria infection, only asnH mutant showed deficiency in their ability to enhance host innate immunity, and complementation of the mutation by introduction of the wild-type asnH in the mutant strain recovered the immuno-augmenting activity. AsnH protein exhibited asparagine synthetase activity when the lysozyme-treated cell wall extracts of L. casei ATCC 27139 was added as substrate. The asnH mutants lost the thick and rigid peptidoglycan features that are characteristic to the wild-type cells, indicating that AsnH of L. casei is involved in peptidoglycan biosynthesis. These results indicate that asnH is required for the construction of the peptidoglycan composition involved in the immune-activating capacity of L. casei ATCC 27139.
Asunto(s)
Aspartatoamoníaco Ligasa/genética , Elementos Transponibles de ADN/genética , Inmunidad/genética , Lacticaseibacillus casei/enzimología , Lacticaseibacillus casei/genética , Mutagénesis Insercional/genética , Probióticos/metabolismo , Secuencia de Aminoácidos , Animales , Aspartatoamoníaco Ligasa/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Bacteriófagos/fisiología , Pared Celular/metabolismo , Citocinas/metabolismo , Disacáridos/química , Disacáridos/metabolismo , Genes Bacterianos , Lacticaseibacillus casei/citología , Lacticaseibacillus casei/inmunología , Listeriosis/microbiología , Listeriosis/patología , Metilnitronitrosoguanidina , Ratones , Datos de Secuencia Molecular , Mutación/genética , Peptidoglicano/química , Peptidoglicano/metabolismo , Alineación de Secuencia , Bazo/metabolismo , Bazo/microbiologíaRESUMEN
In common bean, asparagine synthetase (AS; EC 6.3.5.4) is encoded by three members of a multigene family called PVAS1, PVAS2 and PVAS3. Two of these genes, PVAS1 and PVAS2, have been extensively studied, but little is known about PVAS3, remaining unclear whether PVAS3 function is redundant to the other AS or if it plays a specific role in Phaseolus vulgaris metabolism. In this work, we used a molecular approach to characterize PVAS3 expression and to gain some knowledge about its physiological function. We showed that, in contrast to PVAS1 and PVAS2, PVAS3 was expressed in all organs analyzed. Interestingly, PVAS3 was the AS gene most highly expressed in nodules, leaves and pods at the earliest stages of development, and its expression decreased as these organs developed. Expression of PVAS3 parallels the accumulation of AS protein and the asparagine content during the earliest stages of nodule, leaf and pod development, suggesting an important role for PVAS3 in the synthesis of asparagine in that period. Furthermore, PVAS3 was not repressed by light, as most class-II AS genes. Surprisingly, fertilization of nodulated plants with nitrate or ammonium, conditions that induce PVAS1 and PVAS2 and the shift from ureides to amide synthesis, repressed the expression of PVAS3 in nodules and roots. The possible implications of this regulation are discussed.
Asunto(s)
Aspartatoamoníaco Ligasa/genética , Regulación de la Expresión Génica de las Plantas , Phaseolus/genética , Proteínas de Plantas/genética , Asparagina/biosíntesis , Aspartatoamoníaco Ligasa/química , Aspartatoamoníaco Ligasa/metabolismo , Nitrógeno/farmacología , Phaseolus/enzimología , Phaseolus/crecimiento & desarrollo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismoRESUMEN
Asparagine synthetase A (AsnA) catalyzes asparagine synthesis using aspartate, ATP, and ammonia as substrates. Asparagine is formed in two steps: the ß-carboxylate group of aspartate is first activated by ATP to form an aminoacyl-AMP before its amidation by a nucleophilic attack with an ammonium ion. Interestingly, this mechanism of amino acid activation resembles that used by aminoacyl-tRNA synthetases, which first activate the α-carboxylate group of the amino acid to form also an aminoacyl-AMP before they transfer the activated amino acid onto the cognate tRNA. In a previous investigation, we have shown that the open reading frame of Pyrococcus abyssi annotated as asparaginyl-tRNA synthetase (AsnRS) 2 is, in fact, an archaeal asparagine synthetase A (AS-AR) that evolved from an ancestral aspartyl-tRNA synthetase (AspRS). We present here the crystal structure of this AS-AR. The fold of this protein is similar to that of bacterial AsnA and resembles the catalytic cores of AspRS and AsnRS. The high-resolution structures of AS-AR associated with its substrates and end-products help to understand the reaction mechanism of asparagine formation and release. A comparison of the catalytic core of AS-AR with those of archaeal AspRS and AsnRS and with that of bacterial AsnA reveals a strong conservation. This study uncovers how the active site of the ancestral AspRS rearranged throughout evolution to transform an enzyme activating the α-carboxylate group into an enzyme that is able to activate the ß-carboxylate group of aspartate, which can react with ammonia instead of tRNA.
Asunto(s)
Aspartatoamoníaco Ligasa/química , Pyrococcus abyssi/enzimología , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Amoníaco/química , Amoníaco/metabolismo , Asparagina/química , Asparagina/metabolismo , Aspartatoamoníaco Ligasa/metabolismo , Aspartato-ARNt Ligasa/química , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Evolución Molecular , Modelos Moleculares , Estructura Terciaria de Proteína , Pyrococcus abyssi/química , Aminoacil-ARN de Transferencia/químicaRESUMEN
Four previously identified maize asparagine synthetase (AsnS) genes and a soy AsnS gene have been cloned and expressed in Escherichia coli. The enzymes have been purified and kinetically characterized. The plant AsnS proteins were expressed mainly in the inclusion bodies although small amounts of one form (ZmAsnS2) were recovered in the soluble fraction. In order to measure the kinetic properties of these enzymes a sensitive assay based on the detection of Asn by HPLC has been developed. In addition a method to refold the recombinant plant AsnS to produce active enzyme has been developed. The plant AsnS enzymes are kinetically distinct with substantial differences in K(m) (Gln) and V(max) values when compared to each other. These differences may be important factors for transgenic studies using AsnS genes for crop improvement.
Asunto(s)
Aspartatoamoníaco Ligasa/farmacocinética , Glycine max/enzimología , Proteínas de Plantas/química , Zea mays/enzimología , Aspartatoamoníaco Ligasa/química , Aspartatoamoníaco Ligasa/genética , Cromatografía Líquida de Alta Presión , Cuerpos de Inclusión , Isoenzimas/química , Pliegue de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacocinética , SolubilidadRESUMEN
Fredericamycin (FDM) A is a pentadecaketide natural product that features an amide linkage. Analysis of the fdm cluster from Streptomyces griseus ATCC 43944, however, failed to reveal genes encoding the types of amide synthetases commonly seen in natural product biosynthesis. Here, we report in vivo and in vitro characterizations of FdmV, an asparagine synthetase (AS) B-like protein, as an amide synthetase that catalyzes the amide bond formation in FDM A biosynthesis. This is supported by the findings that (i) inactivation of fdmV in vivo afforded the ΔfdmV mutant strain SB4027 that abolished FDM A and FDM E production but accumulated FDM C, a biosynthetic intermediate devoid of the characteristic amide linkage; (ii) FdmV in vitro catalyzes conversion of FDM C to FDM B, a known intermediate for FDM A biosynthesis (apparent K(m) = 162 ± 67 µM and k(cat) = 0.11 ± 0.02 min(-1)); and (iii) FdmV also catalyzes the amidation of FDM M-3, a structural analog of FDM C, to afford amide FDM M-6 in vitro, albeit at significantly reduced efficiency. Preliminary enzymatic studies revealed that, in addition to the common nitrogen sources (L-Gln and free amine) of class II glutamine amidotransferases (to which AS B belongs), FdmV can also utilize L-Asn as a nitrogen donor. The amide bond formation in FDM A biosynthesis is proposed to occur after C-8 hydroxylation but before the carbaspirocycle formation.
Asunto(s)
Amida Sintasas/química , Amidas/química , Regulación Bacteriana de la Expresión Génica , Streptomyces griseus/metabolismo , Asparagina/química , Aspartatoamoníaco Ligasa/química , Catálisis , Dominio Catalítico , Hidroxilación , Isoquinolinas/metabolismo , Cinética , Modelos Químicos , Mutación , Nitrógeno/química , Proteínas Recombinantes/química , Compuestos de Espiro/metabolismoRESUMEN
Inhibitor docking studies have implicated a conserved glutamate residue (Glu-348) as a general base in the synthetase active site of the enzyme asparagine synthetase B from Escherichia coli (AS-B). We now report steady-state kinetic, isotope transfer, and positional isotope exchange experiments for a series of site-directed AS-B mutants in which Glu-348 is substituted by conservative amino acid replacements. We find that formation of the ß-aspartyl-AMP intermediate, and therefore the eventual production of asparagine, is dependent on the presence of a carboxylate side chain at this position in the synthetase active site. In addition, Glu-348 may also play a role in mediating the conformational changes needed to (i) coordinate, albeit weakly, the glutaminase and synthetase activities of the enzyme and (ii) establish the structural integrity of the intramolecular tunnel along which ammonia is translocated. The importance of Glu-348 in mediating acyl-adenylate formation contrasts with the functional role of the cognate residues in ß-lactam synthetase (BLS) and carbapenem synthetase (CPS) (Tyr-348 and Tyr-345, respectively), which both likely evolved from asparagine synthetase. Given the similarity of the chemistry catalyzed by AS-B, BLS, and CPS, our work highlights the difficulty of predicting the functional outcome of single site mutations on enzymes that catalyze almost identical chemical transformations.
Asunto(s)
Adenosina Monofosfato/análogos & derivados , Aspartatoamoníaco Ligasa/química , Ácido Aspártico/análogos & derivados , Ácido Glutámico , Adenosina Monofosfato/biosíntesis , Amoníaco/metabolismo , Ácido Aspártico/biosíntesis , Dominio Catalítico , Proteínas de Escherichia coli/química , Cinética , Mutagénesis Sitio-DirigidaRESUMEN
The first sulfoximine-based inhibitor of human asparagine synthetase (ASNS) with nanomolar potency has been shown to suppress proliferation of asparaginase-resistant MOLT-4 cells in the presence of L-asparaginase. This validates literature hypotheses concerning the viability of human ASNS as a target for new drugs against acute lymphoblastic leukemia and ovarian cancer. Developing structure-function relationships for this class of human ASNS inhibitors has proven difficult, however, primarily because of the absence of rapid synthetic procedures for constructing highly functionalized sulfoximines. We now report conditions for the efficient preparation of these compounds by coupling sulfoxides and sulfamides in the presence of a rhodium catalyst. Access to this methodology has permitted the construction of two new adenylated sulfoximines, which were expected to exhibit similar binding affinity and better bioavailability than the original human ASNS inhibitor. Steady-state kinetic characterization of these compounds, however, has revealed the importance of a localized negative charge on the inhibitor that mimics that of the phosphate group in a key acyl-adenylate reaction intermediate. These experiments place an important constraint on the design of sulfoximine libraries for screening experiments to obtain ASNS inhibitors with increased potency and bioavailability.
Asunto(s)
Aspartatoamoníaco Ligasa/antagonistas & inhibidores , Aspartatoamoníaco Ligasa/metabolismo , Metionina Sulfoximina/análogos & derivados , Metionina Sulfoximina/farmacología , Aspartatoamoníaco Ligasa/química , Catálisis , Humanos , Metionina Sulfoximina/síntesis química , Modelos Moleculares , Estructura Molecular , Conformación Proteica , Rodio/química , Sulfonamidas/química , Sulfóxidos/químicaRESUMEN
Beta-lactam-synthesizing enzymes carbapenam synthetase (CPS) and beta-lactam synthetase (beta-LS) are evolutionarily linked to a common ancestor, asparagine synthetase B (AS-B). These three relatives catalyze substrate acyl-adenylation and nucleophilic acyl substitution by either an external (AS-B) or internal (CPS, beta-LS) nitrogen source. Unlike AS-B, crystal structures of CPS and beta-LS revealed a putative Tyr-Glu dyad (CPS, Y345/E380; beta-LS, Y348/E382) proposed to deprotonate the respective internal nucleophile. CPS and beta-LS site-directed mutagenesis (Y345/8A, Y345/8F, E380/2D, E380/2Q, E380A) resulted in the reduction of their catalytic efficiency, with Y345A, E380A, and E382Q producing undetectable amounts of beta-lactam product. However, [(32)P]PP(i)-ATP exchange assays demonstrated Y345A and E380A undergo the first half-reaction, with the remaining active mutants showing decreased forward commitment to beta-lactam cyclization. pH-rate profiles of CPS and beta-LS supported the importance of a Tyr-Glu dyad in beta-lactam formation and suggested its reverse protonation in beta-LS. The kinetics of CPS double-site mutants reinforced the synergism of Tyr-Glu in catalysis. Furthermore, significant solvent isotope effects on k(cat) ((D)k(cat)) for Y345F (1.9) and Y348F (1.7) maintained the assignment of Y345/8 in proton transfer. A proton inventory on Y348F determined its (D)(k(cat)/K(m)) = 0.2 to arise from multiple reactant-state fractionation factors, presumably from water molecule(s) replacing the missing Tyr hydroxyl. The role of a CPS and beta-LS Tyr-Glu catalytic dyad was solidified by a significant decrease in mutant k(cat) viscosity dependence with respect to the wild-type enzymes. The evolutionary relation and potential for engineered biosynthesis were demonstrated by beta-LS acting as a carbapenam synthetase.