Secondary literature sources for Adenylsucc_synt
The following references were automatically generated.
- Eaazhisai K et al.
- Crystal structure of fully ligated adenylosuccinate synthetase from Plasmodium falciparum.
- J Mol Biol. 2004; 335: 1251-64
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In the absence of the de novo purine nucleotide biosynthetic pathway in parasitic protozoa, purine salvage is of primary importance for parasite survival. Enzymes of the salvage pathway are, therefore, good targets for anti-parasitic drugs. Adenylosuccinate synthetase (AdSS), catalysing the first committed step in the synthesis of AMP from IMP, is a potential target for anti-protozoal chemotherapy. We report here the crystal structure of adenylosuccinate synthetase from the malaria parasite, Plasmodium falciparum, complexed to 6-phosphoryl IMP, GDP, Mg2+ and the aspartate analogue, hadacidin at 2 A resolution. The overall architecture of P. falciparum AdSS (PfAdSS) is similar to the known structures from Escherichia coli, mouse and plants. Differences in substrate interactions seen in this structure provide a plausible explanation for the kinetic differences between PfAdSS and the enzyme from other species. Additional hydrogen bonding interactions of the protein with GDP may account for the ordered binding of substrates to the enzyme. The dimer interface of PfAdSS is also different, with a pronounced excess of positively charged residues. Differences highlighted here provide a basis for the design of species-specific inhibitors of the enzyme.
- Sivaraman J, Li Y, Banks J, Cane DE, Matte A, Cygler M
- Crystal structure of Escherichia coli PdxA, an enzyme involved in the pyridoxal phosphate biosynthesis pathway.
- J Biol Chem. 2003; 278: 43682-90
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Pyridoxal 5'-phosphate is an essential cofactor for many enzymes responsible for the metabolic conversions of amino acids. Two pathways for its de novo synthesis are known. The pathway utilized by Escherichia coli consists of six enzymatic steps catalyzed by six different enzymes. The fourth step is catalyzed by 4-hydroxythreonine-4-phosphate dehydrogenase (PdxA, E.C. 22.214.171.1242), which converts 4-hydroxy-l-threonine phosphate (HTP) to 3-amino-2-oxopropyl phosphate. This divalent metal ion-dependent enzyme has a strict requirement for the phosphate ester form of the substrate HTP, but can utilize either NADP+ or NAD+ as redox cofactor. We report the crystal structure of E. coli PdxA and its complex with HTP and Zn2+. The protein forms tightly bound dimers. Each monomer has an alpha/beta/alpha-fold and can be divided into two subdomains. The active site is located at the dimer interface, within a cleft between the two subdomains and involves residues from both monomers. A Zn2+ ion is bound within each active site, coordinated by three conserved histidine residues from both monomers. In addition two conserved amino acids, Asp247 and Asp267, play a role in maintaining integrity of the active site. The substrate is anchored to the enzyme by the interactions of its phospho group and by coordination of the amino and hydroxyl groups by the Zn2+ ion. PdxA is structurally similar to, but limited in sequence similarity with isocitrate dehydrogenase and isopropylmalate dehydrogenase. These structural similarities and the comparison with a NADP-bound isocitrate dehydrogenase suggest that the cofactor binding mode of PdxA is very similar to that of the other two enzymes and that PdxA catalyzes a stepwise oxidative decarboxylation of the substrate HTP.
- Borza T, Iancu CV, Pike E, Honzatko RB, Fromm HJ
- Variations in the response of mouse isozymes of adenylosuccinate synthetase to inhibitors of physiological relevance.
- J Biol Chem. 2003; 278: 6673-9
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Vertebrates have acidic and basic isozymes of adenylosuccinate synthetase, which participate in the first committed step of de novo AMP biosynthesis and/or the purine nucleotide cycle. These isozymes differ in their kinetic properties and N-leader sequences, and their regulation may vary with tissue type. Recombinant acidic and basic synthetases from mouse, in the presence of active site ligands, behave in analytical ultracentrifugation as dimers. Active site ligands enhance thermal stability of both isozymes. Truncated forms of both isozymes retain the kinetic parameters and the oligomerization status of the full-length proteins. AMP potently inhibits the acidic isozyme competitively with respect to IMP. In contrast, AMP weakly inhibits the basic isozyme noncompetitively with respect to all substrates. IMP inhibition of the acidic isozyme is competitive, and that of the basic isozyme noncompetitive, with respect to GTP. Fructose 1,6-bisphosphate potently inhibits both isozymes competitively with respect to IMP but becomes noncompetitive at saturating substrate concentrations. The above, coupled with structural information, suggests antagonistic interactions between the active sites of the basic isozyme, whereas active sites of the acidic isozyme seem functionally independent. Fructose 1,6-bisphosphate and IMP together may be dynamic regulators of the basic isozyme in muscle, causing potent inhibition of the synthetase under conditions of high AMP deaminase activity.
- Hou Z, Wang W, Fromm HJ, Honzatko RB
- IMP Alone Organizes the Active Site of Adenylosuccinate Synthetase from Escherichia coli.
- J Biol Chem. 2002; 277: 5970-6
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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.
- Iancu CV, Borza T, Fromm HJ, Honzatko RB
- IMP, GTP, and 6-phosphoryl-IMP complexes of recombinant mouse muscle adenylosuccinate synthetase.
- J Biol Chem. 2002; 277: 26779-87
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Prokaryotes have a single form of adenylosuccinate synthetase that controls the committed step of AMP biosynthesis, but vertebrates have two isozymes of the synthetase. The basic isozyme, which predominates in muscle, participates in the purine nucleotide cycle, has an active site conformation different from that of the Escherichia coli enzyme, and exhibits significant differences in ligand recognition. Crystalline complexes presented here of the recombinant basic isozyme from mouse show the following. GTP alone binds to the active site without inducing a conformational change. IMP in combination with an acetate anion induces major conformational changes and organizes the active site for catalysis. IMP, in the absence of GTP, binds to the GTP pocket of the synthetase. The combination of GTP and IMP results in the formation of a stable complex of 6-phosphoryl-IMP and GDP in the presence or absence of hadacidin. The response of the basic isozyme to GTP alone differs from that of synthetases from plants, and yet the conformation of the mouse basic and E. coli synthetases in their complexes with GDP, 6-phosphoryl-IMP, and hadacidin are nearly identical. Hence, reported differences in ligand recognition among synthetases probably arise from conformational variations observed in partially ligated enzymes.
- Lovering AL, Hyde EI, Searle PF, White SA
- The structure of Escherichia coli nitroreductase complexed with nicotinic acid: three crystal forms at 1.7 A, 1.8 A and 2.4 A resolution.
- J Mol Biol. 2001; 309: 203-13
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Escherichia coli nitroreductase is a flavoprotein that reduces a variety of quinone and nitroaromatic substrates. Its ability to convert relatively non-toxic prodrugs such as CB1954 (5-[aziridin-1-yl]-2,4-dinitrobenzamide) into highly cytotoxic derivatives has led to interest in its potential for cancer gene therapy. We have determined the structure of the enzyme bound to a substrate analogue, nicotinic acid, from three crystal forms at resolutions of 1.7 A, 1.8 A and 2.4 A, representing ten non-crystallographically related monomers. The enzyme is dimeric, and has a large hydrophobic core; each half of the molecule consists of a five-stranded beta-sheet surrounded by alpha-helices. Helices F and F protrude from the core region of each monomer. There is an extensive dimer interface, and the 15 C-terminal residues extend around the opposing monomer, contributing the fifth beta-strand. The active sites lie on opposite sides of the molecule, in solvent-exposed clefts at the dimer interface. The FMN forms hydrogen bonds to one monomer and hydrophobic contacts to both; its si face is buried. The nicotinic acid stacks between the re face of the FMN and Phe124 in helix F, with only one hydrogen bond to the protein. If the nicotinamide ring of the coenzyme NAD(P)H were in the same position as that of the nicotinic acid ligand, its C4 atom would be optimally positioned for direct hydride transfer to flavin N5. Comparison of the structure with unliganded flavin reductase and NTR suggests reduced mobility of helices E and F upon ligand binding. Analysis of the structure explains the broad substrate specificity of the enzyme, and provides the basis for rational design of novel prodrugs and for site-directed mutagenesis for improved enzyme activity.
- Trapani S, Linss J, Goldenberg S, Fischer H, Craievich AF, Oliva G
- Crystal structure of the dimeric phosphoenolpyruvate carboxykinase (PEPCK) from Trypanosoma cruzi at 2 A resolution.
- J Mol Biol. 2001; 313: 1059-72
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ATP-dependent phosphoenolpyruvate carboxykinase (PEPCK) (ATP: oxaloacetate carboxylyase (transphosphorylating), EC 126.96.36.199) is a key enzyme involved in the catabolism of glucose and amino acids in the parasite Trypanosoma cruzi, the causative agent of Chagas' disease. Due to the significant differences in the amino acid sequence and substrate specificity of the human enzyme (PEPCK (GTP-dependent), EC 188.8.131.52), the parasite enzyme has been considered a good target for the development of new anti-chagasic drugs. We have solved the crystal structure of the recombinant PEPCK of T. cruzi up to 2.0 A resolution, characterised the dimeric organisation of the enzyme by solution small angle X-ray scattering (SAXS) and compared the enzyme structure with the known crystal structure of the monomeric PEPCK from Escherichia coli. The dimeric structure possesses 2-fold symmetry, with each monomer sharing a high degree of structural similarity with the monomeric structure of the E. coli PEPCK. Each monomer folds into two complex mixed alpha/beta domains, with the active site located in a deep cleft between the domains. The two active sites in the dimer are far apart from each other, in an arrangement that seems to permit an independent access of the substrates to the two active sites. All residues of the E. coli PEPCK structure that had been found to interact with substrates and metal cofactors have been found conserved and in a substantially equivalent spatial disposition in the T. cruzi PEPCK structure. No substrate or metal ion was present in the crystal structure. A sulphate ion from the crystallisation medium has been found bound to the active site. Solution SAXS data suggest that, in solutions with lower sulphate concentration than that used for the crystallisation experiments, the actual enzyme conformation may be slightly different from its conformation in the crystal structure. This could be due to a conformational transition upon sulphate binding, similar to the ATP-induced transition observed in the E. coli PEPCK, or to crystal packing effects. The present structure of the T. cruzi PEPCK will provide a good basis for the modelling of new anti-chagasic drug leads.
- Lemke CT, Howell PL
- The 1.6 A crystal structure of E. coli argininosuccinate synthetase suggests a conformational change during catalysis.
- Structure. 2001; 9: 1153-64
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BACKGROUND: Argininosuccinate synthetase (AS) is the rate-limiting enzyme of both the urea and arginine-citrulline cycles. In mammals, deficiency of AS leads to citrullinemia, a debilitating and often fatal autosomal recessive urea cycle disorder, whereas its overexpression for sustained nitric oxide production via the arginine-citrulline cycle leads to the potentially fatal hypotension associated with septic and cytokine-induced circulatory shock. RESULTS: The crystal structure of E. coli AS (EAS) has been determined by the use of selenomethionine incorporation and MAD phasing. The structure has been refined at 1.6 A resolution in the absence of its substrates and at 2.0 A in the presence of aspartate and citrulline (EAS*CIT+ASP). Each monomer of this tetrameric protein has two structural domains: a nucleotide binding domain similar to that of the "N-type" ATP pyrophosphatase class of enzymes, and a novel catalytic/multimerization domain. The EAS*CIT+ASP structure clearly describes the binding of citrulline at the cleft between the two domains and of aspartate to a loop of the nucleotide binding domain, whereas homology modeling with the N-type ATP pyrophosphatases has provided the location of ATP binding. CONCLUSIONS: The first three-dimensional structures of AS are reported. The fold of the nucleotide binding domain confirms AS as the fourth structurally defined member of the N-type ATP pyrophosphatases. The structures identify catalytically important residues and suggest the requirement for a conformational change during the catalytic cycle. Sequence similarity between the bacterial and human enzymes has been used for providing insight into the structural and functional effects of observed clinical mutations.
- Iancu CV, Borza T, Choe JY, Fromm HJ, Honzatko RB
- Recombinant mouse muscle adenylosuccinate synthetase: overexpression, kinetics, and crystal structure.
- J Biol Chem. 2001; 276: 42146-52
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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.
- Moy FJ et al.
- NMR structure of free RGS4 reveals an induced conformational change upon binding Galpha.
- Biochemistry. 2000; 39: 7063-73
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Heterotrimeric guanine nucleotide-binding proteins (G-proteins) are transducers in many cellular transmembrane signaling systems where regulators of G-protein signaling (RGS) act as attenuators of the G-protein signal cascade by binding to the Galpha subunit of G-proteins (G(i)(alpha)(1)) and increasing the rate of GTP hydrolysis. The high-resolution solution structure of free RGS4 has been determined using two-dimensional and three-dimensional heteronuclear NMR spectroscopy. A total of 30 structures were calculated by means of hybrid distance geometry-simulated annealing using a total of 2871 experimental NMR restraints. The atomic rms distribution about the mean coordinate positions for residues 5-134 for the 30 structures is 0.47 +/- 0.05 A for the backbone atoms, 0. 86 +/- 0.05 A for all atoms, and 0.56 +/- 0.04 A for all atoms excluding disordered side chains. The NMR solution structure of free RGS4 suggests a significant conformational change upon binding G(i)(alpha)(1) as evident by the backbone atomic rms difference of 1. 94 A between the free and bound forms of RGS4. The underlying cause of this structural change is a perturbation in the secondary structure elements in the vicinity of the G(i)(alpha)(1) binding site. A kink in the helix between residues K116-Y119 is more pronounced in the RGS4-G(i)(alpha)(1) X-ray structure relative to the free RGS4 NMR structure, resulting in a reorganization of the packing of the N-terminal and C-terminal helices. The presence of the helical disruption in the RGS4-G(i)(alpha)(1) X-ray structure allows for the formation of a hydrogen-bonding network within the binding pocket for G(i)(alpha)(1) on RGS4, where RGS4 residues D117, S118, and R121 interact with residue T182 from G(i)(alpha)(1). The binding pocket for G(i)(alpha)(1) on RGS4 is larger and more accessible in the free RGS4 NMR structure and does not present the preformed binding site observed in the RGS4-G(i)(alpha)(1) X-ray structure. This observation implies that the successful complex formation between RGS4 and G(i)(alpha)(1) is dependent on both the formation of the bound RGS4 conformation and the proper orientation of T182 from G(i)(alpha)(1). The observed changes for the free RGS4 NMR structure suggest a mechanism for its selectivity for the Galpha-GTP-Mg(2+) complex and a means to facilitate the GTPase cycle.
- Safo MK et al.
- X-ray structure of Escherichia coli pyridoxine 5'-phosphate oxidase complexed with FMN at 1.8 A resolution.
- Structure. 2000; 8: 751-62
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BACKGROUND: Escherichia coli pyridoxine 5'-phosphate oxidase (PNPOx) catalyzes the terminal step in the biosynthesis of pyridoxal 5'-phosphate (PLP), a cofactor used by many enzymes involved in amino acid metabolism. The enzyme oxidizes either the 4'-hydroxyl group of pyridoxine 5'-phosphate (PNP) or the 4'-primary amine of pyridoxamine 5'-phosphate (PMP) to an aldehyde. PNPOx is a homodimeric enzyme with one flavin mononucleotide (FMN) molecule non-covalently bound to each subunit. A high degree of sequence homology among the 15 known members of the PNPOx family suggests that all members of this group have similar three-dimensional folds. RESULTS: The crystal structure of PNPOx from E. coli has been determined to 1.8 A resolution. The monomeric subunit folds into an eight-stranded beta sheet surrounded by five alpha-helical structures. Two monomers related by a twofold axis interact extensively along one-half of each monomer to form the dimer. There are two clefts at the dimer interface that are symmetry-related and extend from the top to the bottom of the dimer. An FMN cofactor that makes interactions with both subunits is located in each of these two clefts. CONCLUSIONS: The structure is quite similar to the recently deposited 2.7 A structure of Saccharomyces cerevisiae PNPOx and also, remarkably, shares a common structural fold with the FMN-binding protein from Desulfovibrio vulgaris and a domain of chymotrypsin. This high-resolution E. coli PNPOx structure permits predictions to be made about residues involved in substrate binding and catalysis. These predictions provide testable hypotheses, which can be answered by making site-directed mutants.
- Jeffery CJ, Gloss LM, Petsko GA, Ringe D
- The role of residues outside the active site: structural basis for function of C191 mutants of Escherichia coli aspartate aminotransferase.
- Protein Eng. 2000; 13: 105-12
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In previous kinetic studies of Escherichia coli aspartate aminotransferase, it was determined that some substitutions of conserved cysteine 191, which is located outside of the active site, altered the kinetic parameters of the enzyme (Gloss,L.M., Spencer,D. E. and Kirsch,J.F., 1996, Protein Struct. Funct. Genet., 24, 195-208). The mutations resulted in an alkaline shift of 0.6-0.8 pH units for the pK(a) of the internal aldimine between the PLP cofactor and Lys258. The change in the pK(a) affected the pH dependence of the k(cat)/K(m) (aspartate) values for the mutant enzymes. To help to understand these observations, crystal structures of five mutant forms of E.coli aspartate aminotransferase (the maleate complexes of C191S, C191F, C191Y and C191W, and C191S without maleate) were determined at about 2 A resolution in the presence of the pyridoxal phosphate cofactor. The overall three-dimensional fold of each mutant enzyme is the same as that of the wild-type protein, but there is a rotation of the mutated side chain around its C(alpha)-C(beta) bond. This side chain rotation results in a change in the pattern of hydrogen bonding connecting the mutant residue and the protonated Schiff base of the cofactor, which could account for the altered pK(a) of the Schiff base imine nitrogen that was reported previously. These results demonstrate how residues outside the active site can be important in helping determine the subtleties of the active site amino acid geometries and interactions and how mutations outside the active site can have effects on catalysis. In addition, these results help explain the surprising result previously reported that, for some mutant proteins, replacement of a buried cysteine with an aromatic side chain did not destabilize the protein fold. Instead, rotation around the C(alpha)-C(beta) bond allowed each large aromatic side chain to become buried in a nearby pocket without large changes in the enzyme's backbone geometry.
- Thompson TB, Garrett JB, Taylor EA, Meganathan R, Gerlt JA, Rayment I
- Evolution of enzymatic activity in the enolase superfamily: structure of o-succinylbenzoate synthase from Escherichia coli in complex with Mg2+ and o-succinylbenzoate.
- Biochemistry. 2000; 39: 10662-76
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The X-ray structures of the ligand free (apo) and the Mg(2+)*o-succinylbenzoate (OSB) product complex of o-succinylbenzoate synthase (OSBS) from Escherichia coli have been solved to 1.65 and 1.77 A resolution, respectively. The structure of apo OSBS was solved by multiple isomorphous replacement in space group P2(1)2(1)2(1); the structure of the complex with Mg(2+)*OSB was solved by molecular replacement in space group P2(1)2(1)2. The two domain fold found for OSBS is similar to those found for other members of the enolase superfamily: a mixed alpha/beta capping domain formed from segments at the N- and C-termini of the polypeptide and a larger (beta/alpha)(7)beta barrel domain. Two regions of disorder were found in the structure of apo OSBS: (i) the loop between the first two beta-strands in the alpha/beta domain; and (ii) the first sheet-helix pair in the barrel domain. These regions are ordered in the product complex with Mg(2+)*OSB. As expected, the Mg(2+)*OSB pair is bound at the C-terminal end of the barrel domain. The electron density for the phenyl succinate component of the product is well-defined; however, the 1-carboxylate appears to adopt multiple conformations. The metal is octahedrally coordinated by Asp(161), Glu(190), and Asp(213), two water molecules, and one oxygen of the benzoate carboxylate group of OSB. The loop between the first two beta-strands in the alpha/beta motif interacts with the aromatic ring of OSB. Lys(133) and Lys(235) are positioned to function as acid/base catalysts in the dehydration reaction. Few hydrogen bonding or electrostatic interactions are involved in the binding of OSB to the active site; instead, most of the interactions between OSB and the protein are either indirect via water molecules or via hydrophobic interactions. As a result, evolution of both the shape and the volume of the active site should be subject to few structural constraints. This would provide a structural strategy for the evolution of new catalytic activities in homologues of OSBS and a likely explanation for how the OSBS from Amycolaptosis also can catalyze the racemization of N-acylamino acids [Palmer, D. R., Garrett, J. B., Sharma, V., Meganathan, R., Babbitt, P. C., and Gerlt, J. A. (1999) Biochemistry 38, 4252-4258].
- Choe JY, Poland BW, Fromm HJ, Honzatko RB
- Mechanistic implications from crystalline complexes of wild-type and mutant adenylosuccinate synthetases from Escherichia coli.
- Biochemistry. 1999; 38: 6953-61
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Asp13 and His41 are essential residues of adenylosuccinate synthetase, putatively catalyzing the formation of adenylosuccinate from an intermediate of 6-phosphoryl-IMP. Wild-type adenylosuccinate synthetase and three mutant synthetases (Arg143 --> Leu, Lys16 --> Gln, and Arg303 --> Leu) from Eschericha coli have been crystallized in the presence of IMP, hadacidin (an analogue of L-aspartate), Mg2+, and GTP. The active site of each complex contains 6-phosphoryl-IMP, Mg2+, GDP, and hadacidin, except for the Arg303 --> Leu mutant, which does not bind hadacidin. In response to the formation of 6-phosphoryl-IMP, Asp13 enters the inner coordination sphere of the active site Mg2+. His41 hydrogen bonds with 6-phosphoryl-IMP, except in the Arg303 --> Leu complex, where it remains bound to the guanine nucleotide. Hence, recognition of the active site Mg2+ by Asp13 evidently occurs after the formation of 6-phosphoryl-IMP, but recognition of the intermediate by His41 may require the association of L-aspartate with the active site. Structures reported here support a mechanism in which Asp13 and His41 act as the catalytic base and acid, respectively, in the formation of 6-phosphoryl-IMP, and then act together as catalytic acids in the subsequent formation of adenylosuccinate.
- Hou Z, Cashel M, Fromm HJ, Honzatko RB
- Effectors of the stringent response target the active site of Escherichia coli adenylosuccinate synthetase.
- J Biol Chem. 1999; 274: 17505-10
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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.
- Honzatko RB, Fromm HJ
- Structure-function studies of adenylosuccinate synthetase from Escherichia coli.
- Arch Biochem Biophys. 1999; 370: 1-8
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Adenylosuccinate synthetase catalyzes the first committed step in the de novo biosynthesis of AMP, thermodynamically coupling the hydrolysis of GTP to the formation of adenylosuccinate from l-aspartate and IMP. The enzyme from Esherichia coli undergoes a ligand-induced dimerization, which leads to the assembly of a complete active site. The binding of IMP causes conformational changes over distances of 30 A, the end result of which is the activation of essential catalytic elements and the organization of the binding pocket for Mg(2+)-GTP. The enzyme promotes first a phosphoryl transfer from GTP to the 6-oxygen atom of IMP, by way of a transition state that has characteristics of both associative and dissociative reaction pathways. Following the formation of 6-phosphoryl-IMP, the enzyme then catalyzes the nucleophilic displacement of the 6-phosphoryl group by the alpha-amino group of l-aspartate in a transition state, which requires two metal cations.
- Lee P, Gorrell A, Fromm HJ, Colman RF
- Implication of arginine-131 and arginine-303 in the substrate site of adenylosuccinate synthetase of Escherichia coli by affinity labeling with 6-(4-bromo-2,3-dioxobutyl)thioadenosine 5'-monophosphate.
- Biochemistry. 1999; 38: 5754-63
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Adenylosuccinate synthetase from Escherichia coli is inactivated in a biphasic reaction by 6-(4-bromo-2,3-dioxobutyl)thioadenosine 5'-monophosphate (6-BDB-TAMP) at pH 7.0 and 25 degrees C. The initial fast-phase inactivation is not affected by the presence of active-site ligands and can be completely eliminated by blocking Cys291 of the enzyme with N-ethylmaleimide (NEM). Reaction of the NEM-treated enzyme with 6-BDB-[32P]TAMP results in 2 mol of reagent incorporated/mol of enzyme subunit. The inactivation kinetics of the slow-phase exhibit an apparent KI of 40.6 microM and kmax of 0.0228 min-1. Active-site ligands, either adenylosuccinate or IMP and GTP, completely prevent inactivation of the enzyme by 6-BDB-TAMP, whereas IMP or IMP and aspartate is much less effective in protection. 6-BDB-TAMP-inactivated enzyme has a 3-fold increase in Km for aspartate with no change in Km for IMP or GTP. Protease digestion of 6-BDB-[32P]TAMP inactivated enzyme reveals that both Arg131 and Arg303 are modified by the affinity-labeling reagent. The crystal structure [Poland, B. W., Fromm, H. J., and Honzatko, R. B. (1996) J. Mol. Biol. 264, 1013-1027] and site-directed mutagenesis [Kang, C., Sun, N., Poland, B. W., Gorrell, A., and Fromm, H. J. (1997) J. Biol. Chem. 272, 11881-11885] of E. coli adenylosuccinate synthetase show that Arg303 interacts with the carboxyl group of aspartate and the 2'-OH of the ribose of IMP and Arg131 is involved in stabilizing aspartate in the active site of the enzyme. We conclude that 6-BDB-TAMP functions as a reactive adenylosuccinate analogue in modifying both Arg131 and Arg303 in the active site of adenylosuccinate synthetase.
- Kang C, Sun N, Poland BW, Gorrell A, Honzatko RB, Fromm HJ
- Residues essential for catalysis and stability of the active site of Escherichia coli adenylosuccinate synthetase as revealed by directed mutation and kinetics.
- J Biol Chem. 1997; 272: 11881-5
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Examined here by directed mutation, circular dichroism spectroscopy, and kinetics are the relationships of five residues, Asp13, Glu14, Lys16, His41, and Arg131, to the catalytic function and structural organization of adenylosuccinate synthetase from Escherichia coli. The D13A mutant has no measurable activity. Mutants E14A and H41N exhibit 1% of the activity of the wild-type enzyme and 2-7-fold increases in the Km of substrates. The mutant K16Q has 34% of the activity of wild-type enzyme and Km values for substrates virtually unchanged from those of the wild-type system. Mutation of Arg131 to leucine caused only a 4-fold increase in the Km for aspartate relative to the wild-type enzyme. The dramatic effects of the D13A, E14A, and H41N mutations on kcat are consistent with the putative roles assigned to Asp13 (catalytic base), His41 (catalytic acid), and Glu14 (structural organization of the active site). The modest effect of the R131L mutation on the binding of aspartate is also in harmony with recent crystallographic investigations, which suggests that Arg131 stabilizes the conformation of the loop that binds the beta-carboxylate of aspartate. The modest effect of the K16Q mutation, however, contrasts with significant changes brought about by the mutation of the corresponding lysines in the P-loop of other GTP- and ATP-binding proteins. Crystallographic structures place Lys16 in a position of direct interaction with the gamma-phosphate of GTP. Furthermore, lysine is present at corresponding positions in all known sequences of adenylosuccinate synthetase. We suggest that along with a modest role in stabilizing the transition state of the phosphotransfer reaction, Lys16 may stabilize the enzyme structurally. In addition, the modest loss of catalytic activity of the K16Q mutant may confer such a selective disadvantage to E. coli that this seemingly innocuous mutation is not tolerated in nature.
- Wang W, Gorrell A, Honzatko RB, Fromm HJ
- A study of Escherichia coli adenylosuccinate synthetase association states and the interface residues of the homodimer.
- J Biol Chem. 1997; 272: 7078-84
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The state of aggregation of adenylosuccinate synthetase from Escherichia coli is a point of controversy, with crystal structures indicating a dimer and some solution studies indicating a monomer. Crystal structures implicate Arg143 and Asp231 in stabilizing the dimer, with Arg143 interacting directly with bound IMP of the 2-fold related subunit. Residue Arg143 was changed to Lys and Leu, and residue Asp231 was changed to Ala. Matrix-assisted laser desorption ionization mass spectroscopy and analytical ultracentrifugation of the wild-type and the mutant enzymes indicate a mixture of monomers and dimers, with a majority of the enzyme in the monomeric state. In the presence of active site ligands, the wild-type enzyme exists almost exclusively as a dimer, whereas the mutant enzymes show only slightly decreased dissociation constants for the dimerization. Initial rate kinetic studies of the wild-type and mutant enzymes show similar kcat and Km values for aspartate. However, increases in the Km values of GTP and IMP are observed for the mutant. Changes in dissociation constants for IMP are comparable with changes in Km values. Our results suggest that IMP binding induces enzyme dimerization and that two residues in the interface region, Arg143 and Asp231, play significant roles in IMP and GTP binding.
- Poland BW, Bruns C, Fromm HJ, Honzatko RB
- Entrapment of 6-thiophosphoryl-IMP in the active site of crystalline adenylosuccinate synthetase from Escherichia coli.
- J Biol Chem. 1997; 272: 15200-5
- Display abstract
Crystal structures of adenylosuccinate synthetase from Escherichia coli complexed with Mg2+, 6-thiophosphoryl-IMP, GDP, and hadacidin at 298 and 100 K have been refined to R-factors of 0.171 and 0.206 against data to 2.8 and 2.5 A resolution, respectively. Interactions of GDP, Mg2+ and hadacidin are similar to those observed for the same ligands in the complex of IMP, GDP, NO3-, Mg2+ and hadacidin (Poland, B. W., Fromm, H. J. & Honzatko, R. B. (1996). J. Mol. Biol. 264, 1013-1027). Although crystals were grown from solutions containing 6-mercapto-IMP and GTP, the electron density at the active site is consistent with 6-thiophosphoryl-IMP and GDP. Asp-13 and Gln-224 probably work in concert to stabilize the 6-thioanion of 6-mercapto-IMP, which in turn is the nucleophile in the displacement of GDP from the gamma-phosphate of GTP. Once formed, 6-thiophosphoryl-IMP is stable in the active site of the enzyme under the conditions of the structural investigation. The direct observation of 6-thiophosphoryl-IMP in the active site is consistent with the putative generation of 6-phosphoryl-IMP along the reaction pathway of the synthetase.
- Wang W, Hou Z, Honzatko RB, Fromm HJ
- Relationship of conserved residues in the IMP binding site to substrate recognition and catalysis in Escherichia coli adenylosuccinate synthetase.
- J Biol Chem. 1997; 272: 16911-6
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Gln34, Gln224, Leu228, and Ser240 are conserved residues in the vicinity of bound IMP in the crystal structure of Escherichia coli adenylosuccinate synthetase. Directed mutations were carried out, and wild-type and mutant enzymes were purified to homogeneity. Circular dichroism spectroscopy indicated no difference in secondary structure between the mutants and the wild-type enzyme in the absence of substrates. Mutants L228A and S240A exhibited modest changes in their initial rate kinetics relative to the wild-type enzyme, suggesting that neither Leu228 nor Ser240 play essential roles in substrate binding or catalysis. The mutants Q224M and Q224E exhibited no significant change in KmGTP and KmASP and modest changes in KmIMP relative to the wild-type enzyme. However, kcat decreased 13-fold for the Q224M mutant and 10(4)-fold for the Q224E mutant relative to the wild-type enzyme. Furthermore, the Q224E mutant showed an optimum pH at 6.2, which is 1.5 pH units lower than that of the wild-type enzyme. Tryptophan emission fluorescence spectra of Q224M, Q224E, and wild-type proteins under denaturing conditions indicate comparable stabilities. Mutant Q34E exhibits a 60-fold decrease in kcat compared with that of the wild-type enzyme, which is attributed to the disruption of the Gln34 to Gln224 hydrogen bond observed in crystal structures. Presented here is a mechanism for the synthetase, whereby Gln224 works in concert with Asp13 to stabilize the 6-oxyanion of IMP.
- Sticht H, Gallert KC, Krauss G, Rosch P
- Homology modeling of adenylosuccinate synthetase from Saccharomyces cerevisiae reveals a possible binding region for single-stranded ARS sequences.
- J Biomol Struct Dyn. 1997; 14: 667-75
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Adenylosuccinate synthetase from Saccharomyces cerevisiae was investigated in order to find a structural explanation for its ability to bind specifically to single-stranded ARS elements (autonomously replicating sequences). Using the E. coli enzyme as template, a model for the structure of adenylosuccinate synthetase from S. cerevisiae was generated and subsequently refined by molecular dynamics techniques. The resulting three-dimensional structure offers an explanation for the DNA binding activity of the yeast enzyme by revealing a distinct basic region that is not present in the homologous enzymes from other organisms. The model is also in good agreement with biochemical data available for a mutant protein in which Glycine 252 is replaced by Aspartate. On the basis of the model a significant structural distortion near the catalytic center was predicted for this mutant, corresponding well to the enzymatic inactivity observed. The mutant enzyme shows larger structural fluctuations than the wild-type protein according to the results of two independent molecular dynamics simulations.
- Poland BW et al.
- Refined crystal structure of adenylosuccinate synthetase from Escherichia coli complexed with hydantocidin 5'-phosphate, GDP, HPO4(2-), Mg2+, and hadacidin.
- Biochemistry. 1996; 35: 15753-9
- Display abstract
A crystal structure of adenylosuccinate synthetase from Escherichia coli, complexed with 5'-phosphate, GDP, HPO4(2-), Mg2+, and hadacidin at 100 K, has been refined to an Rfactor of 0.195 against data to 2.6 A resolution. Bond lengths and angles deviate from expected values by 0.012 A and 1.86 degrees, respectively. Lys 16 and backbone amides 15-17 and 42 interact with the phosphates of GDP, while Ser 414, Asp 333, and backbone amides 331 and 416 interact with the base. Mg2+ is octahedrally coordinated. Oxygen atoms from GDP, phosphate, and hadacidin define the equatorial plane of coordination of the Mg2+, while backbone carbonyl 40 and the side chain of Asp 13 are the apical ligands. HPO4(2-) hydrogen bonds with Lys 16, His 41, backbone amides 13, 40, and 224, and the base moiety of the hydantocidin inhibitor. The carboxylate of hadacidin interacts with Arg 303 and Thr 301; its N-formyl group coordinates to Mg2+, and its hydroxyl group hydrogen bonds with Asp 13. The 5'-phosphate of the hydantocidin inhibitor interacts with Asn 38, Thr 129, and Thr 239 but is approximately 3.5 A from Arg 143 (related by molecular 2-fold symmetry). The base moiety of hydantocidin 5'-phosphate hydrogen bonds to Gln 224 and participates in a hydrogen-bonded network that includes the phosphate molecule, several water molecules, and Asp 13. Hydantocidin 5'-phosphate, GDP, HPO4(2-), and Mg2+ may represent a set of synergistic inhibitors even more effective than the combination of IMP, GDP, NO3-, and Mg2+.
- Poland BW, Fromm HJ, Honzatko RB
- Crystal structures of adenylosuccinate synthetase from Escherichia coli complexed with GDP, IMP hadacidin, NO3-, and Mg2+.
- J Mol Biol. 1996; 264: 1013-27
- Display abstract
Crystal structures of adenylosuccinate synthetase from Esherichia coli complexed with Mg2+, IMP, GDP, NO3- and hadacidin at 298 and 100 K have been refined to R-factors of 0.188 and 0.206 against data to 2.8 A and 2.5 A resolution, respectively. Conformational changes of up to 9 A relative to the unligated enzyme occur in loops that bind to Mg2+, GDP, IMP and hadacidin. Mg2+ binds directly to GDP, NO3-, hadacidin and the protein, but is only five-coordinated. Asp13, which approaches, but does not occupy the sixth coordination site of Mg2+, hydrogen bonds to N1 of IMP. The nitrogen atom of NO3- is approximately 2.7 A from O6 of IMP, reflecting a strong electrostatic interaction between the electron-deficient nitrogen atom and the electron-rich O6. The spatial relationships between GDP, NO3- and Mg2+ suggest an interaction between the beta,gamma-bridging oxygen atom of GTP and Mg2+ in the enzyme-substrate complex. His41 hydrogen bonds to the beta-phosphate group of GDP and approaches bound NO3-. The aldehyde group of hadacidin coordinates to the Mg2+, while its carboxyl group interacts with backbone amide groups 299 to 303 and the side-chain of Arg303. The 5'-phosphate group of IMP interacts with Asn38, Thr129, Thr239 and Arg143 (from a monomer related by 2-fold symmetry). A mechanism is proposed for the two-step reaction governed by the synthetase, in which His41 and Asp13 are essential catalytic side-chains.
- Louie GV et al.
- The three-dimensional structure of Escherichia coli porphobilinogen deaminase at 1.76-A resolution.
- Proteins. 1996; 25: 48-78
- Display abstract
Porphobilinogen deaminase (PBGD) catalyses the polymerization of four molecules of porphobilinogen to form the 1-hydroxymethylbilane, preuroporphyrinogen, a key intermediate in the biosynthesis of tetrapyrroles. The three-dimensional structure of wild-type PBGD from Escherichia coli has been determined by multiple isomorphous replacement and refined to a crystallographic R-factor of 0.188 at 1.76 A resolution. the polypeptide chain of PBGD is folded into three alpha/beta domains. Domains 1 and 2 have a similar overall topology, based on a five-stranded, mixed beta-sheet. These two domains, which are linked by two hinge segments but otherwise make few direct interactions, form an extensive active site cleft at their interface. Domain 3, an open-faced, anti-parallel sheet of three strands, interacts approximately equally with the other two domains. The dipyrromethane cofactor is covalently attached to a cysteine side-chain borne on a flexible loop of domain 3. The cofactor serves as a primer for the assembly of the tetrapyrrole product and is held within the active site cleft by hydrogen-bonds and salt-bridges that are formed between its acetate and propionate side-groups and the polypeptide chain. The structure of a variant of PBGD, in which the methionines have been replaced with selenomethionines, has also been determined. The cofactor, in the native and functional form of the enzyme, adopts a conformation in which the second pyrrole ring (C2) occupies an internal position in the active site cleft. On oxidation, however, this C2 ring of the cofactor adopts a more external position that may correspond approximately to the site of substrate binding and polypyrrole chain elongation. The side-chain of Asp84 hydrogen-bonds the hydrogen atoms of both cofactor pyrrole nitrogens and also potentially the hydrogen atom of the pyrrole nitrogen of the porphobilinogen molecule bound to the proposed substrate binding site. This group has a key catalytic role, possibly in stabilizing the positive charges that develop on the pyrrole nitrogens during the ring-coupling reactions. Possible mechanisms for the processive elongation of the polypyrrole chain involve: accommodation of the elongating chain within the active site cleft, coupled with shifts in the relative positions of domains 1 and 2 to carry the terminal ring into the appropriate position at the catalytic site; or sequential translocation of the elongating polypyrrole chain, attached to the cofactor on domain 3, through the active site cleft by the progressive movement of domain 3 with respect to domains 1 and 2. Other mechanisms are considered although the amino acid sequence comparisons between PBGDs from all species suggest they share the same three-dimensional structure and mechanism of activity.
- Scapin G, Reddy SG, Blanchard JS
- Three-dimensional structure of meso-diaminopimelic acid dehydrogenase from Corynebacterium glutamicum.
- Biochemistry. 1996; 35: 13540-51
- Display abstract
Diaminopimelate dehydrogenase catalyzes the NADPH-dependent reduction of ammonia and L-2-amino-6-ketopimelate to form meso-diaminopimelate, the direct precursor of L-lysine in the bacterial lysine biosynthetic pathway. Since mammals lack this metabolic pathway inhibitors of enzymes in this pathway may be useful as antibiotics or herbicides. Diaminopimelate dehydrogenase catalyzes the only oxidative deamination of an amino acid of D configuration and must additionally distinguish between two chiral amino acid centers on the same symmetric substrate. The Corynebacterium glutamicum enzyme has been cloned, expressed in Escherichia coli, and purified to homogeneity using standard biochemical procedures [Reddy, S. G., Scapin, G., & Blanchard, J. S. (1996) Proteins: Structure, Funct. Genet. 25, 514-516]. The three-dimensional structure of the binary complex of diaminopimelate dehydrogenase with NADP+ has been solved using multiple isomorphous replacement procedures and noncrystallographic symmetry averaging. The resulting model has been refined against 2.2 A diffraction data to a conventional crystallographic R-factor of 17.0%. Diaminopimelate dehydrogenase is a homodimer of structurally not identical subunits. Each subunit is composed of three domains. The N-terminal domain contains a modified dinucleotide binding domain, or Rossman fold (six central beta-strands in a 213456 topology surrounded by five alpha-helices). The second domain contains two alpha-helices and three beta-strands. This domain is referred to as the dimerization domain, since it is involved in forming the monomer--monomer interface of the dimer. The third or C-terminal domain is composed of six beta-strands and five alpha-helices. The relative position of the N- and C-terminal domain in the two monomers is different, defining an open and a closed conformation that may represent the enzyme's binding and active state, respectively. In both monomers the nucleotide is bound in an extended conformation across the C-terminal portion of the beta-sheet of the Rossman fold, with its C4 facing the C-terminal domain. In the closed conformer two molecules of acetate have been refined in this region, and we postulate that they define the DAP binding site. The structure of diaminopimelate dehydrogenase shows interesting similarities to the structure of glutamate dehydrogenase [Baker, P. J., Britton, K. L., Rice, D. W., Rob, A., & Stillmann, T.J. (1992a) J. Mol. Biol. 228, 662-671] and leucine dehydrogenase [Baker, P.J., Turnbull, A.P., Sedelnikova, S.E., Stillman, T. J., & Rice, D. W. (1995) Structure 3, 693-705] and also resembles the structure of dihydrodipicolinate reductase [Scapin, G., Blanchard, J. S., & Sacchettini, J. C. (1995) Biochemistry 34, 3502-3512], the enzyme immediately preceding it in the diaminopimelic acid/lysine biosynthetic pathway.
- Poland BW, Hou Z, Bruns C, Fromm HJ, Honzatko RB
- Refined crystal structures of guanine nucleotide complexes of adenylosuccinate synthetase from Escherichia coli.
- J Biol Chem. 1996; 271: 15407-13
- Display abstract
Structures of adenylosuccinate synthetase from Escherichia coli complexed with guanosine-5'-(beta,gamma-imido) triphosphate and guanosine-5'-(beta,gamma-methylene)triphosphate in the presence and the absence of Mg2+ have been refined to R-factors below 0.2 against data to a nominal resolution of 2.7 A. Asp333 of the synthetase hydrogen bonds to the exocyclic 2-amino and endocyclic N1 groups of the guanine nucleotide base, whereas the hydroxyl of Ser414 and the backbone amide of Lys331 hydrogen bond to the 6-oxo position. The side chains of Lys331 and Pro417 pack against opposite faces of the guanine nucleotide base. The synthetase recognizes neither the N7 position of guanine nucleotides nor the ribose group. Electron density for the guanine-5'-(beta,gamma-imido) triphosphate complex is consistent with a mixture of the triphosphate nucleoside and its hydrolyzed diphosphate nucleoside bound to the active site. The base, ribose, and alpha-phosphate positions overlap, but the beta-phosphates occupy different binding sites. The binding of guanosine-5'-(beta,gamma-methylene)triphosphate to the active site is comparable with that of guanosine-5'-(beta, gamma-imido)triphosphate. No electron density, however, for the corresponding diphosphate nucleoside is observed. In addition, electron density for bound Mg2+ is absent in these nucleotide complexes. The guanine nucleotide complexes of the synthetase are compared with complexes of other GTP-binding proteins and to a preliminary structure of the complex of GDP, IMP, Mg2+, and succinate with the synthetase. The enzyme, under conditions reported here, does not undergo a conformational change in response to the binding of guanine nucleotides, and minimally IMP and/or Mg2+ must be present in order to facilitate the complete recognition of the guanine nucleotide by the synthetase.
- Kang C, Kim S, Fromm HJ
- Subunit complementation of Escherichia coli adenylosuccinate synthetase.
- J Biol Chem. 1996; 271: 29722-8
- Display abstract
Data are presented, based upon subunit complementation experiments, that suggest that Escherichia coli adenylosuccinate synthetase contains two shared active sites between its dimeric interface. This conclusion was alluded to by use of mutant forms of adenylosuccinate synthetase previously prepared by site-directed mutagenesis. The experiments indicate that, although the R143L and D13A mutants have low or no activity independently, when they are mixed, a significant amount of activity was obtained. These results indicate that the subunits exchange with each other to form heterodimers with a single viable wild-type active site. The kcat value for the active hybrid active site in the R143L-D13A heterodimer is virtually identical to that observed with the wild-type enzyme, and the other kinetic parameters are very similar to those found for the wild-type enzyme. An analysis of the restoration of the activity in the presence of substrates suggests that GTP and IMP stabilize the dimeric structure of the protein. A comparison of the restoration of the activity using different combinations of mutants provides evidence indicating that some of the GTP binding elements, including the P-loop, in the protein are important for subunit integrity. Also, for the first time, a comprehensive analysis of subunit complementation is performed for the two inactive mutants (R143L and D13A) where the dissociation constants for the R143L-D13A heterodimer and the D13A homodimer were determined to be 21 and 2.9 microM, respectively. A concentration dependence of the specific activity of the wild-type protein in this study shows that the Kd for dimer dissociation is approximately 1 microM.
- Moe OA, Baker-Malcolm JF, Wang W, Kang C, Fromm HJ, Colman RF
- Involvement of arginine 143 in nucleotide substrate binding at the active site of adenylosuccinate synthetase from Escherichia coli.
- Biochemistry. 1996; 35: 9024-33
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Adenylosuccinate synthetase from Escherichia coli is inactivated in a biphasic reaction by guanosine 5'-O-[S-(4-bromo-2,3-dioxobutyl)thio]phosphate (GMPSBDB) at pH 7.1 and 25 degrees C. Reaction of the enzyme with [8-3H]GMPSBDB results in the incorporation of 2 mol of the reagent/mol of subunit; in the presence of active site ligands the incorporation is reduced to 1 mol of reagent/mol of subunit. GMPSBDB reacts with Cys-291 in the initial rapid reaction which is accompanied by loss of 50% of the enzymatic activity; this reaction is not affected by the presence of active site ligands. In the slower reaction, GMPSBDB inactivates the enzyme by reacting with Arg-143. The inactivation kinetics of the slower phase are consistent with the formation of an enzyme--GMPSBDB complex having a Kd of 42 microM. Active site nucleotides, either adenylosuccinate or IMP + GTP, prevent both slower phase inactivation and labeling of Arg-143. Replacement of Arg-143 with a Leu by site-directed mutagenesis does not change the catalytic constant or the K(m) for aspartate but does significantly impair nucleotide binding: the Michaelis constants for IMP and GTP increase by 60-fold and 10-fold, respectively, in the R143L mutant. The crystal structure of the E. coli enzyme [Poland, B.W., Silva, M.M., Serra, M.A., Cho, Y., Kim, K. H., Harris, E.M.S., & Honzatko, R.B. (1993) J. Biol. Chem. 268, 25334--25342] shows that Arg-143 from one subunit projects into the putative active site of the other subunit. These results indicate that both subunits of dimeric adenylosuccinate synthetase contribute to each active site and that Arg-143 plays an important role in nucleotide binding.
- Wang W, Poland BW, Honzatko RB, Fromm HJ
- Identification of arginine residues in the putative L-aspartate binding site of Escherichia coli adenylosuccinate synthetase.
- J Biol Chem. 1995; 270: 13160-3
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Three arginine residues in the putative aspartate binding site of Escherichia coli adenylosuccinate synthetase were changed to leucines by site-directed mutagenesis. The mutant enzymes R303L, R304L, and R305L were purified to homogeneity on the basis of sodium dodecyl sulfate polyacrylamide gel electrophoresis and characterized by CD spectrometry and initial rate kinetics. CD spectral analysis indicated no differences in secondary structure between the mutants and the wild-type enzyme in the absence of substrates. The Km values for GTP and IMP for the mutants and the wild-type enzyme were comparable. However, the mutant enzymes exhibited 50-200-fold increases in their values of Km for the substrate aspartate relative to the wild-type enzyme. Although the kcat values for the mutant enzymes decreased, the changes were not as dramatic as those observed for the Km of aspartate. The modeling of aspartate in the crystal structure of the complex of adenylosuccinate synthetase with IMP and MgGDP-1 is consistent with the results of mutagenesis, placing the alpha- and beta-carboxylates of aspartate near the side chains of Arg-131, -303, and -305.
- Silva MM, Poland BW, Hoffman CR, Fromm HJ, Honzatko RB
- Refined crystal structures of unligated adenylosuccinate synthetase from Escherichia coli.
- J Mol Biol. 1995; 254: 431-46
- Display abstract
Crystal structures of unligated adenylosuccinate synthetase from Escherichia coli in space groups P2(1) and P2(1)2(1)2(1) have been refined to R-factors of 0.199 and 0.206 against data to 2.0 and 2.5 A, respectively. Bond lengths and angles deviate from expected values by 0.011 A and 1.7 degrees for the P2(1) crystal form and by 0.015 A and 1.7 degrees for the P2(1)2(1)2(1) crystal form. The fold of the polypeptide chain is dominated by a central beta-sheet, which is composed of nine parallel strands and a tenth antiparallel strand. Extending off from this central beta-sheet are four subdomains. The four subdomains contribute loops of residues that are disordered or have high thermal parameters. At least three of these loops (residues 42 to 52, 120 to 131 and 298 to 304) contribute essential residues to the putative active site of the synthetase. In the absence of ligands, much of the active site of the synthetase exists in an ill-defined conformational state. Two, nearly independent regions contribute residues to the interface between polypeptide chains of the synthetase dimer. A pair of helices (H4 and H5) interact with their symmetry-equivalent mates by way of residues that are not conserved amongst the known sequences of the synthetase. The second interface region involves conserved residues belonging to structural elements that connect strands of the central beta-sheet. Residues putatively involved in the binding of IMP lie at or near the interface between polypeptide chains of the dimer. Of the four sequence elements putatively common to all GTP hydrolases, the synthetase has only the guanine recognition element and a glycine-rich loop (P-loop). Although the base recognition element is essentially identical with those of the p21 ras and G alpha proteins, the P-loop of the synthetase is extended in size relative to the P-loops of other GTP hydrolases. The P-loop has two acid residues (Asp13 and Glu14), which are found in the P-loops of only the synthetase family. Glu14 may be involved in the stabilization of the enlarged P-loop of the synthetase, whereas Asp13 may play a role in catalysis and in the coordination of Mg2+. The structural elements of the p21 ras and G alpha proteins responsible for binding Mg2+ are either absent from the synthetase or unavailable for the coordination of metal cations.
- Kang C, Fromm HJ
- Characterization of the putative GTP-binding site residues of Escherichia coli adenylosuccinate synthetase by site-directed mutagenesis.
- Arch Biochem Biophys. 1994; 310: 475-80
- Display abstract
Adenylosuccinate synthetase contains amino acid sequences in its GTP-binding domain that are homologous to other G-proteins. This homology includes a glycine-rich phosphate-binding loop, GXXXXGK, and a guanine-specific binding region, (N/T/Q)KXD; however, virtually no other sequence homology exists between other G-proteins and adenylosuccinate synthetase. On the basis of X-ray diffraction studies, the folding topologies of the synthetase and the p21 ras proteins are different. Yet, residues that interact with GTP in the p21 ras proteins are present in the synthetase in nearly identical positions. We chose therefore to study the G15V mutant, a phosphate-binding loop mutant, and K331L and K331R, two mutants of Lys331 that are involved in guanine ring binding. The Km values for GTP of adenylosuccinate synthetase mutants, K331L and K331R, when compared to those of the wild-type enzyme, were 27- and 20-fold increased, respectively, without any significant change in the Km values for IMP. Because both mutations affected the Km values for GTP similarly, whereas the kcat and secondary structure were essentially unchanged, it is suggested that Lys331 is located in the GTP-binding site of adenylosuccinate synthetase and the terminal N zeta of the Lys is not necessarily important in GTP-binding on the enzyme. Therefore, Lys331 may interact with GTP through hydrophobic interactions between its linear side chain and the aromatic ring of the guanine base of GTP. Also, structural characterization of the G15V mutant was carried out using circular dichroism (CD) spectrometry, NMR spectroscopy, and spectrofluorometry. The CD spectral data indicated that the secondary structure of the G15V mutant was significantly altered by GTP and IMP, whereas that of the wild-type enzyme is not changed; however, the two enzymes exhibited similar secondary structures in the absence of substrates. The NMR spectra of both enzymes were also similar in the absence of substrates. The dissociation constant (Kd) for IMP of the G15V mutant was 4.8-fold larger than its Km value which was 1.5-fold increased compared to the wild-type enzyme. From these findings, it was concluded that the phosphate-binding region of adenylosuccinate synthetase is involved in a conformational change induced by GTP and IMP binding, and that GTP and IMP binding depend on the presence of the other substrate at the active site of the enzyme. These results suggest that the Lys331 of adenylosuccinate synthetase may play similar roles in the function and structure to that of GTP-binding proteins.(ABSTRACT TRUNCATED AT 400 WORDS)
- Kang C, Sun N, Honzatko RB, Fromm HJ
- Replacement of Asp333 with Asn by site-directed mutagenesis changes the substrate specificity of Escherichia coli adenylosuccinate synthetase from guanosine 5'-triphosphate to xanthosine 5'-triphosphate.
- J Biol Chem. 1994; 269: 24046-9
- Display abstract
The aspartate residue of the (N/T)KXD concensus sequence for GTP-binding proteins is present in the eight available sequences of adenylosuccinate synthetase. Reported here is a comprehensive analysis of the substrate specificity of mutant enzymes, where the conserved Asp333 of the synthetase from Escherichia coli is changed to asparagine, glutamate, and glutamine by site-directed mutagenesis. The mutants D333N, D333E, and D333Q generally show decreased kcat values and increased Km values for GTP. The decreased values of kcat exhibited by the mutants indicate that the interactions between Asp333 and the guanine are relayed by some mechanism to the catalytic residues around the gamma-phosphate of GTP, and that the energy provided by the interaction between Asp333 and the guanine moiety of GTP is utilized for rearrangement of the catalytic residues. The three mutants each have higher affinity for xanthosine 5'-triphosphate (XTP) and ITP than does the wild-type enzyme. In fact, the D333N mutant uses XTP more effectively than the wild-type enzyme employs GTP as a substrate. The side-chain of Asp333 forms hydrogen bonds with the N-1 and the exocyclic amino group of the guanine base of GTP. In the D333N mutant, this interaction is probably replaced by hydrogen bonds between the amide side chain of Asn333 and N-1 and the 2-oxo group of XTP. The D333Q mutant can use UTP as a substrate more effectively than the wild-type enzyme. The longer side chain of glutamine at residue 333 favors pyrimidine nucleotides over the purine nucleotides, GTP, XTP, and ITP. These results demonstrate that Asp333 in the (N/T)KXD consensus sequence of adenylosuccinate synthetase from E. coli is a determinant for GTP-specificity.
- Liu F, Dong Q, Fromm HJ
- Site-directed mutagenesis of the phosphate-binding consensus sequence in Escherichia coli adenylosuccinate synthetase.
- J Biol Chem. 1992; 267: 2388-92
- Display abstract
Adenylosuccinate synthetases from different sources contain an N-terminal glycine-rich sequence GDEGKGK, which is homologous to the conserved sequence GXXXXGK found in many other guanine nucleotide-binding proteins or enzymes. To determine the role of this sequence in the structure and function of Escherichia coli adenylosuccinate synthetase, site-directed mutagenesis was performed to generate five mutant enzymes: G12V (Gly12----Val), G15V (Gly15----Val), G17V (Gly17----Val), K18R (Lys18----Arg), and I19T (Ile19----Thr). Comparison of the kinetic properties of the wild-type enzyme and those of the mutant enzymes revealed that the sequence is critical for enzyme activity. Replacement of Gly12, Gly15, or Gly17 with Val, or replacement of Lys18 with Arg, resulted in significant decreases in the kcat/Km values of the enzyme. Because the consensus sequence GXXXXGK(T/S) has been found in many GTP-binding proteins, isoleucine at position 19 in the E. coli adenylosuccinate synthetase was changed to threonine to produce the sequence GDEGKGKT. This mutation, which more closely resembles the consensus sequence, resulted in a 160-fold increase in the Km value for substrate GTP; however, there were no great changes for the other two substrates, IMP and aspartate. Based on these data, we suggest that the N-terminal glycinerich sequence in E. coli adenylosuccinate synthetase plays a more important role in enzyme catalysis than in substrate binding. In addition, a hydrophobic amino acid residue such as isoleucine, leucine, or valine, rather than threonine, may play a critical role in GTP binding in adenosuccinate synthetase. These findings suggest that the glycine-rich sequence in adenylosuccinate synthetase functions differently relative to those in other GTP binding proteins or enzymes.
- Wiesmuller L, Wittbrodt J, Noegel AA, Schleicher M
- Purification and cDNA-derived sequence of adenylosuccinate synthetase from Dictyostelium discoideum.
- J Biol Chem. 1991; 266: 2480-5
- Display abstract
Adenylosuccinate synthetase (IMP:L-aspartate ligase (GDP), EC 184.108.40.206) plays an important role in purine biosynthesis catalyzing the GTP-dependent conversion of IMP to AMP. The enzyme was purified from the cytosol of Dictyostelium discoideum using GTP-agarose chromatography as the critical step. It has an apparent molecular mass of 44 kDa. Monoclonal antibodies identified several forms of the enzyme with pI values between 8.1 and 9.0. Michaelis-Menten constants (Km) were low for the nucleotide substrates IMP (Km = 30 microM) and GTP (Km = 35 microM) as compared with the value for aspartic acid (Km = 440 microM). These values are in good agreement with constants reported from other organisms. Immunological studies indicated that the protein is predominantly localized in the cytosol and only partially associated with particulate fractions. The enzyme is present throughout the developmental cycle of D. discoideum. Using monoclonal antibodies, the gene was cloned from a lambda gt11 expression library. The complete sequence represents the first reported primary structure of an eucaryotic adenylosuccinate synthetase. Southern blots hybridized with a cDNA probe demonstrate that adenylosuccinate synthetase is encoded by a single gene and contains at least one intron. The deduced amino acid sequence shows 43% identity to adenylosuccinate synthetase from Escherichia coli. Homologous regions include short sequence motifs, such as the glycine-rich loop which is typical for GTP-binding proteins.
- Dong Q, Liu F, Myers AM, Fromm HJ
- Evidence for an arginine residue at the substrate binding site of Escherichia coli adenylosuccinate synthetase as studied by chemical modification and site-directed mutagenesis.
- J Biol Chem. 1991; 266: 12228-33
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Chemical modification of adenylosuccinate synthetase from Escherichia coli with phenylglyoxal resulted in an inhibition of enzyme activity with a second-order rate constant of 13.6 M-1 min-1. The substrates, GTP or IMP, partially protected the enzyme against inactivation by the chemical modification. The other substrate, aspartate, had no such effect even at a high concentration. In the presence of both IMP and GTP during the modification, nearly complete protection of the enzyme against inactivation was observed. Stoichiometry studies with [7-14C]phenylglyoxal showed that only 1 reactive arginine residue was modified by the chemical reagent and that this arginine residue could be shielded by GTP and IMP. Sequence analysis of tryptic peptides indicated that Arg147 is the site of phenylglyoxal chemical modification. This arginine has been changed to leucine by site-directed mutagenesis. The mutant enzyme (R147L) showed increased Michaelis constants for IMP and GTP relative to the wild-type system, whereas the Km for aspartate exhibited a modest decrease as compared with the native enzyme. In addition, kcat of the R147L mutant decreased by a factor of 1.3 x 10(4). On the bases of these observations, it is suggested that Arg147 is critical for enzyme catalysis.
- Soans C, Fromm HJ
- Studies of ligand binding to Escherichia coli adenylosuccinate synthetase.
- Arch Biochem Biophys. 1991; 291: 107-12
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Dissociation constants of Escherichia coli adenylosuccinate synthetase with IMP, GTP, adenylosuccinate, and AMP (a competitive inhibitor for IMP) were determined by measuring the extent of quenching of the intrinsic tryptophan fluorescence of the enzyme. The enzyme has one binding site for each of these ligands. Aspartate and GDP did not quench the fluorescence to any great extent, and their dissociation constants could not be determined. These ligand binding studies were generally supportive of the kinetic mechanism proposed earlier for the enzyme. Cys291 was modified with the fluorescent chromophores N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonate and tetramethylrhodamine maleimide in order to measure enzyme conformational changes attending ligand binding. The excitation and emission spectra of these fluorophores are not altered by the addition of active site binding ligands. TbGTP and TbGDP were used as native reporter groups, and changes in their fluorescence on complexing with the enzyme and various ligands made it possible to detect conformational changes occurring at the active site. Evidence is presented for abortive complexes of the type: enzyme-TbGTP-adenylosuccinate and enzyme-TbGTP-adenylosuccinate-aspartate. These results suggest that the IMP and aspartate binding sites are spatially separated.
- Wolfe SA, Smith JM
- Nucleotide sequence and analysis of the purA gene encoding adenylosuccinate synthetase of Escherichia coli K12.
- J Biol Chem. 1988; 263: 19147-53
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Adenylosuccinate synthetase (EC 220.127.116.11), encoded by the purA gene of Escherichia coli K12, catalyzes the synthesis of adenylosuccinate (SAMP) from IMP, the first committed step in AMP biosynthesis. The E. coli K12 purA gene and flanking DNA was cloned by miniMu-mediated transduction, and the nucleotide sequence was determined. The mature SAMP synthetase subunit, as deduced from the DNA sequence, contains 427 amino acid residues and has a calculated Mr of 47,277. The size of the purA mRNA was determined by Northern blotting to be approximately 1.5 kilobase pairs. The 5'-end of the purA mRNA was identified by primer extension and is located 23 nucleotides upstream of the ATG translational initiation codon. Comparison of the purA control region with the guaBA control region revealed a common region of dyad symmetry which may suggest mutual elements of regulation. The purA control region did not resemble the control regions of the other known pur loci.
- Bass MB, Fromm HJ, Stayton MM
- Overproduction, purification, and characterization of adenylosuccinate synthetase from Escherichia coli.
- Arch Biochem Biophys. 1987; 256: 335-42
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Adenylosuccinate synthetase, encoded by the purA gene of Escherichia coli, catalyzes the first committed step toward AMP in the de novo purine biosynthetic pathway and plays an important role in the interconversion of purines. A 3.2-kb DNA fragment, which carries the purA gene, was cloned into the temperature-inducible, high-copy-number plasmid vector, pMOB45. Upon temperature induction, cells containing this plasmid produce adenylosuccinate synthetase at approximately 40 times the wild-type level. A scheme is presented for the purification of the overproduced adenylosuccinate synthetase to homogeneity in amounts sufficient for studies of its structure and mechanism. The wild-type and the overproduced adenylosuccinate synthetase enzyme preparations were judged to be identical by the following criteria. The amino acid sequence at the N-terminus of the overproduced enzyme proved identical to the corresponding sequence of the wild-type enzyme. Michaelis constants for both the wild-type and overproduced enzyme preparations were the same. And (iii) both proteins shared similar chromatographic behavior and the same mobility during sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Results from size-exclusion chromatography and SDS-polyacrylamide gel electrophoresis suggest that adenylosuccinate synthetase exists as a dimer of identical, 48,000-Da, subunits.
- Cooper BF, Fromm HJ, Rudolph FB
- Isotope exchange at equilibrium studies with rat muscle adenylosuccinate synthetase.
- Biochemistry. 1986; 25: 7323-7
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The kinetic mechanism of rat muscle adenylosuccinate synthetase was studied by determining the rates of isotope exchange at equilibrium. A random sequential binding mechanism was indicated for both the forward and reverse reactions. Aspartate, adenylosuccinate, GDP, and Pi were determined to bind in rapid equilibrium. GTP exchanges with both GDP and Pi at the same rate, which is similar to the exchange rate of IMP with adenylosuccinate. Aspartate exchanges with adenylosuccinate at a higher rate than does IMP over the range of concentrations tested. The slower IMP and GTP exchange rates suggest a forward binding mechanism containing a preferred path in which the quaternary complex is most often formed by aspartate binding to the E-GTP-IMP complex. This preferred path is consistent with an interaction between IMP and GTP in the absence of aspartate as determined by isotope scrambling experiments [Bass, M. B., Fromm, H. J., & Rudolph, F. B. (1984) J. Biol. Chem. 259, 12330-12333]. However, the products of such an interaction are tightly bound to the enzyme as no partial exchange reactions between adenylosuccinate and aspartate in the presence or absence of Pi were detected.
- Stayton MM, Rudolph FB, Fromm HJ
- Regulation, genetics, and properties of adenylosuccinate synthetase: a review.
- Curr Top Cell Regul. 1983; 22: 103-41
- Matsuda Y, Shiraki H, Nakagawa H
- Molecular transformation of tumor adenylosuccinate synthetase and characteristics of its converting factor.
- Cancer Res. 1982; 42: 112-6
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The properties of adenylosuccinate synthetase [inosine monophosphate:L-aspartate ligase (guanosine 5'-diphosphate-forming) EC 18.104.22.168]) of rat Yoshida sarcoma ascites tumor cells changed during purification. The isoelectric points (pI) of the crude and purified enzymes were 5.0 and 5.9, respectively. The Km values of the crude enzyme for inosine monophosphate, aspartate, and guanosine 5'-triphosphate were calculated to be 99 +/- 1 (S.E.), 870 +/- 40, and 27 +/- 2 microM, respectively, while those of the purified enzyme were 410 +/- 10, 980 +/- 50, and 69 +/- 5 microM, respectively. These data indicate that the crude enzyme should be more effective for activity than the purified one. It was found that the change in pI occurred during diethylaminoethyl cellulose column chromatography and that, during this step a compound, named pI-converting factor (ICF), was separated from the enzyme molecule. On addition of ICF, the pI of the purified enzyme changed from 5.9 to 5.0, indicating that the pI conversion was dependent on ICF and was reversible. ICF was nondialyzable, heat stable, and partly precipitated with 1 N perchloric acid but was not affected by 1 N KOH. It was partially degraded by DNAse I. These results suggest that ICF is a DNA-like compound.
- Baugher BW, Montonaro L, Welch MM, Rudolph FB
- Changes in isozymes of adenylosuccinate synthetase.
- Biochem Biophys Res Commun. 1980; 94: 123-9