Combinatorial interactions regulate cardiac expression of the murine adenylosuccinate synthetase 1 gene.
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The mammalian heart begins contracting at the linear tube stage during embryogenesis and continuously pumps, nonstop, throughout the entire lifetime of the animal. Therefore, the cardiac energy metabolizing pathways must be properly established and efficiently functioning. While the biochemistry of these pathways is well defined, limited information regarding the regulation of cardiac metabolic genes is available. Previously, we reported that 1.9 kilobase pairs of murine adenylosuccinate synthetase 1 gene (Adss1) 5'-flanking DNA directs high levels of reporter expression to the adult transgenic heart. In this report, we define the 1.9-kilobase pair fragment as a cardiac-specific enhancer that controls correct spatiotemporal expression of a reporter similar to the endogenous Adss1 gene. A 700-base pair fragment within this region activates a heterologous promoter specifically in adult transgenic hearts. Proteins present in a cardiac nuclear extract interact with potential transcription factor binding sites of this region and these cis-acting sites play important regulatory roles in the cardiac expression of this reporter. Finally, we report that several different cardiac transcription factors trans-activate the 1.9HSCAT construct through these sites and that combinations result in enhanced reporter expression. Adss1 appears to be one of the first target genes identified for the bHLH factors Hand1 and Hand2. (+info)
Effectors of the stringent response target the active site of Escherichia coli adenylosuccinate synthetase.
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Guanosine 5'-diphosphate 3'-diphosphate (ppGpp), a pleiotropic effector of the stringent response, potently inhibits adenylosuccinate synthetase from Escherichia coli as an allosteric effector and/or as a competitive inhibitor with respect to GTP. Crystals of the synthetase grown in the presence of IMP, hadacidin, NO3-, and Mg2+, then soaked with ppGpp, reveal electron density at the GTP pocket which is consistent with guanosine 5'-diphosphate 2':3'-cyclic monophosphate. Unlike ligand complexes of the synthetase involving IMP and GDP, the coordination of Mg2+ in this complex is octahedral with the side chain of Asp13 in the inner sphere of the cation. The cyclic phosphoryl group interacts directly with the side chain of Lys49 and indirectly through bridging water molecules with the side chains of Asn295 and Arg305. The synthetase either directly facilitates the formation of the cyclic nucleotide or scavenges trace amounts of the cyclic nucleotide from solution. Regardless of its mode of generation, the cyclic nucleotide binds far more tightly to the active site than does ppGpp. Conceivably, synthetase activity in vivo during the stringent response may be sensitive to the relative concentrations of several effectors, which together exercise precise control over the de novo synthesis of AMP. (+info)
Adenylosuccinate synthase from Saccharomyces cerevisiae: homologous overexpression, purification and characterization of the recombinant protein.
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Adenylosuccinate synthase (EC 6.3.4.4) catalyses the first committed step in the synthesis of adenosine. We have overexpressed the cloned gene of Saccharomyces cerevisiae (ADE12) in S. cerevisiae. The recombinant enzyme exhibits similar kinetic behaviour to that of the native enzyme purified from S. cerevisiae. This ter-reactant dimeric enzyme shows Michaelis-Menten kinetics only with IMP. l-Aspartate and GTP display a weak negative co-operativity (Hill coefficient 0. 8-0.9). This negative co-operativity has not yet been reported for adenylosuccinate synthases from other organisms. Another unusual feature of the enzyme from S. cerevisiae is its negligible inhibition by adenine nucleotides and its pronounced inhibition by Cl(-) ions. (+info)
Electrical stimulation of neonatal cardiac myocytes activates the NFAT3 and GATA4 pathways and up-regulates the adenylosuccinate synthetase 1 gene.
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Electrically stimulated pacing of cultured cardiomyocytes serves as an experimentally convenient and physiologically relevant in vitro model of cardiac hypertrophy. Electrical pacing triggers a signaling cascade that results in the activation of the muscle-specific Adss1 gene and the repression of the nonmuscle Adss2 isoform. Activation of the Adss1 gene involves the calcineurin-mediated dephosphorylation of NFAT3, allowing its translocation to the nucleus, where it can directly participate in Adss1 gene activation. Mutational studies show that an NFAT binding site located in the Adss1 5'-flanking region is essential for this activation. Electrical pacing also results in the increased synthesis of GATA4, another critical cardiac transcription factor required for Adss1 gene expression. MEF2C also produces transactivation of the Adss1 gene reporter in control and paced cardiac myocytes. Using the Adss1 gene as a model, these studies are the first to demonstrate that electrical pacing activates the calcineurin/NFAT3 and GATA4 pathways as a means of regulating cardiac gene expression. (+info)
Investigation of various genotype characteristics for inosine accumulation in Escherichia coli W3110.
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For the derivation of an inosine-overproducing strain from the wild type microorganism, it is known that the addition of an adenine requirement, removal of purine nucleoside hydrolyzing activity, removal of the feedback inhibition, and repression of key enzymes in the purine nucleotides biosynthetic pathway are essential. Thus, the disruption of purA (adenine requirement), deoD (removal of purine nucleosides phosphorylase activity), purR (derepression of the regulation of purine nucleotides biosynthetic pathway), and the insensitivity of the feedback inhibition of phosphoribosylpyrophosphate (PRPP) amidotransferase by adenosine 5'-monophosphate (AMP) and guanosine 5'-monophosphate (GMP) were done in the Escherichia coli strain W3110, and then the inosine productivity was estimated. In the case of using a plasmid harboring the PRPP amidotransferase gene (purF) that encoded a desensitized PRPP amidotransferase, purF disrupted mutants were used as the host strains. It was found that the innovation of the four genotypes brought about a small amount of inosine accumulation. Furthermore, an adenine auxotrophic mutant of E. coli showed inappropriate adenine use because its growth could not respond efficiently to the concentration of adenine added. As the presence of adenosine deaminase is well known in E. coli and it is thought to be involved in adenine use, a mutant disrupted adenosine deaminase gene (add) was constructed and tested. The mutant, which is deficient in purF, purA, deoD, purR, and add genes, and harboring the desensitized purF as a plasmid, accumulated about 1 g of inosine per liter. Although we investigated the effects of purR disruption and purF gene improvement, unexpectedly an increase in the inosine productivity could not be found with this mutant. (+info)
Vertebrates possess two isozymes of adenylosuccinate synthetase. The acidic isozyme is similar to the synthetase from bacteria and plants, being involved in the de novo biosynthesis of AMP, whereas the basic isozyme participates in the purine nucleotide cycle. Reported here is the first instance of overexpression and crystal structure determination of a basic isozyme of adenylosuccinate synthetase. The recombinant mouse muscle enzyme purified to homogeneity in milligram quantities exhibits a specific activity comparable with that of the rat muscle enzyme isolated from tissue and K(m) parameters for GTP, IMP, and l-aspartate (12, 45, and 140 microm, respectively) similar to those of the enzyme from Escherichia coli. The mouse muscle and E. coli enzymes have similar polypeptide folds, differing primarily in the conformation of loops, involved in substrate recognition and stabilization of the transition state. Residues 65-68 of the muscle isozyme adopt a conformation not observed in any previous synthetase structure. In its new conformation, segment 65-68 forms intramolecular hydrogen bonds with residues essential for the recognition of IMP and, in fact, sterically excludes IMP from the active site. Observed differences in ligand recognition among adenylosuccinate synthetases may be due in part to conformational variations in the IMP pocket of the ligand-free enzymes. (+info)
IMP Alone Organizes the Active Site of Adenylosuccinate Synthetase from Escherichia coli.
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A complete set of substrate/substrate analogs of adenylosuccinate synthetase from Escherichia coli induces dimer formation and a transition from a disordered to an ordered active site. The most striking of the ligand-induced effects is the movement of loop 40-53 by up to 9 A. Crystal structures of the partially ligated synthetase, which either combine IMP and hadacidin or IMP, hadacidin, and Mg(2+)-pyrophosphate, have ordered active sites, comparable with the fully ligated enzyme. More significantly, a crystal structure of the synthetase with IMP alone exhibits a largely ordered active site, which includes the 9 A movement of loop 40-53 but does not include conformational adjustments to backbone carbonyl 40 (Mg(2+) interaction element) and loop 298-304 (L-aspartate binding element). Interactions involving the 5'-phosphoryl group of IMP evidently trigger the formation of salt links some 30 A away. The above provides a structural basis for ligand binding synergism, effects on k(cat) due to mutations far from the site of catalysis, and the complete loss of substrate efficacy due to minor alterations of the 5'-phosphoryl group of IMP. (+info)
Determinants of L-aspartate and IMP recognition in Escherichia coli adenylosuccinate synthetase.
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Adenylosuccinate synthetase governs the first committed step in the de novo synthesis of AMP. Mutations of conserved residues in the synthetase from Escherichia coli reveal significant roles for Val(273) and Thr(300) in the recognition of l-aspartate, even though these residues do not or cannot hydrogen bond with the substrate. The mutation of Thr(300) to alanine increases the K(m) for l-aspartate by 30-fold. In contrast, its mutation to valine causes no more than a 4-fold increase in the K(m) for l-aspartate, while increasing k(cat) by 3-fold. Mutations of Val(273) to alanine, threonine, or asparagine increase the K(m) for l-aspartate from 15- to 40-fold, and concomitantly decrease the K(i) for dicarboxylate analogues of l-aspartate by up to 40-fold. The above perturbations are comparable with those resulting from the elimination of a hydrogen bond between the enzyme and substrate: alanine mutations of Thr(128) and Thr(129) increase the K(m) for IMP by up to 30-fold and the alanine mutation of Thr(301) abolishes catalysis supported by l-aspartate, but has no effect on catalysis supported by hydroxylamine. Structure-based mechanisms, by which the above residues influence substrate recognition, are presented. (+info)