Purification, crystallization and preliminary X-ray analysis of adenylosuccinate synthetase from the fungal pathogen Cryptococcus neoformans.

With increasingly large immunocompromised populations around the world, opportunistic fungal pathogens such as Cryptococcus neoformans are a growing cause of morbidity and mortality. To combat the paucity of antifungal compounds, new drug targets must be investigated. Adenylosuccinate synthetase is a crucial enzyme in the ATP de novo biosynthetic pathway, catalyzing the formation of adenylosuccinate from inosine monophosphate and aspartate. Although the enzyme is ubiquitous and well characterized in other kingdoms, no crystallographic studies on the fungal protein have been performed. Presented here are the expression, purification, crystallization and initial crystallographic analyses of cryptococcal adenylosuccinate synthetase. The crystals had the symmetry of space group P2(1)2(1)2(1) and diffracted to 2.2 Šresolution.

Expression, crystallization and preliminary X-ray crystallographic analysis of alcohol dehydrogenase (ADH) from Kangiella koreensis.

Alcohol dehydrogenases (ADHs) are a group of dehydrogenase enzymes that facilitate the interconversion between alcohols and aldehydes or ketones with the reduction of NAD(+) to NADH. In bacteria, some alcohol dehydrogenases catalyze the opposite reaction as part of fermentation to ensure a constant supply of NAD(+). The adh gene from Kangiella koreensis was cloned and the protein (KkADH) was expressed, purified and crystallized. A KkADH crystal diffracted to 2.5 Šresolution and belonged to the monoclinic space group P2(1), with unit-cell parameters a = 94.1, b = 80.9, c = 115.6 Å, ß = 111.9°. Four monomers were present in the asymmetric unit, with a corresponding VM of 2.55 Å(3) Da(-1) and a solvent content of 51.8%.

Mutational analysis of cysteine 328 and cysteine 368 at the interface of Plasmodium falciparum adenylosuccinate synthetase.

Plasmodium falciparum adenylosuccinate synthetase, a homodimeric enzyme, contains 10 cysteine residues per subunit. Among these, Cys250, Cys328 and Cys368 lie at the dimer interface and are not conserved across organisms. PfAdSS has a positively charged interface with the crystal structure showing additional electron density around Cys328 and Cys368. Biochemical characterization of site directed mutants followed by equilibrium unfolding studies permits elucidation of the role of interface cysteines and positively charged interface in dimer stability. Mutation of interface cysteines, Cys328 and Cys368 to serine, perturbed the monomer-dimer equilibrium in the protein with a small population of monomer being evident in the double mutant. Introduction of negative charge in the form of C328D mutation resulted in stabilization of protein dimer as evident by size exclusion chromatography at high ionic strength buffer and equilibrium unfolding in the presence of urea. These observations suggest that cysteines at the dimer interface of PfAdSS may indeed be charged and exist as thiolate anion.

Overexpression, purification, crystallization and preliminary crystallographic studies of a hyperthermophilic adenylosuccinate synthetase from Pyrococcus horikoshii OT3.

Adenylosuccinate synthetase (AdSS) is a ubiquitous enzyme that catalyzes the first committed step in the conversion of inosine monophosphate (IMP) to adenosine monophosphate (AMP) in the purine-biosynthetic pathway. Although AdSS from the vast majority of organisms is 430-457 amino acids in length, AdSS sequences isolated from thermophilic archaea are 90-120 amino acids shorter. In this study, crystallographic studies of a short AdSS sequence from Pyrococcus horikoshii OT3 (PhAdSS) were performed in order to reveal the unusual structure of AdSS from thermophilic archaea. Crystals of PhAdSS were obtained by the microbatch-under-oil method and X-ray diffraction data were collected to 2.50 Å resolution. The crystal belonged to the trigonal space group P3(2)12, with unit-cell parameters a = b = 57.2, c = 107.9 Å. There was one molecule per asymmetric unit, giving a Matthews coefficient of 2.17 Å(3) Da(-1) and an approximate solvent content of 43%. In contrast, the results of native polyacrylamide gel electrophoresis and analytical ultracentrifugation showed that the recombinant PhAdSS formed a dimer in solution.

Kinetic characterization of adenylosuccinate synthetase from the thermophilic archaea Methanocaldococcus jannaschii.

Adenylosuccinate synthetase (AdSS) catalyzes the Mg2+ dependent condensation of a molecule of IMP with aspartate to form adenylosuccinate, in a reaction driven by the hydrolysis of GTP to GDP. AdSS from the thermophilic archaea, Methanocaldococcus jannaschii (MjAdSS) is 345 amino acids long against an average length of 430-457 amino acids for most mesophilic AdSS. This short AdSS has two large deletions that map to the middle and C-terminus of the protein. This article discusses the detailed kinetic characterization of MjAdSS. Initial velocity and product inhibition studies, carried out at 70 degrees C, suggest a rapid equilibrium random AB steady-state ordered C kinetic mechanism for the MjAdSS catalyzed reaction. AdSS are known to exhibit monomer-dimer equilibrium with the dimer being implicated in catalysis. In contrast, our studies show that MjAdSS is an equilibrium mixture of dimers and tetramers with the tetramer being the catalytically active form. The tetramer dissociates into dimers with a minor increase in ionic strength of the buffer, while the dimer is extremely stable and does not dissociate even at 1.2 M NaCl. Phosphate, a product of the reaction, was found to be a potent inhibitor of MjAdSS showing biphasic inhibition of enzyme activity. The inhibition was competitive with IMP and noncompetitive with GTP. MjAdSS, like the mouse acidic isozyme, exhibits substrate inhibition, with IMP inhibiting enzyme activity at subsaturating GTP concentrations. Regulation of enzyme activity by the glycolytic intermediate, fructose 1,6 bisphosphate, was also observed with the inhibition being competitive with IMP and noncompetitive against GTP.

Adenylosuccinate synthetase of the yeast Saccharomyces cerevisiae: purification and properties.

Adenylosuccinate synthetase (AS-synthetase) was purified from the yeast Saccharomyces cerevisiae. The purification procedure included chromatography on DEAE-cellulose, phosphocellulose, and heparin-agarose. The pH and temperature optima for the enzyme activity (7.0 and 35 degreesC, respectively) and also pH and thermostability of AS-synthetase were determined. The native form of the enzyme exists as a dimer. The Km values for IMP, GTP, and L-aspartate are 1.7, 0.16, and 6.7 mM, respectively. ATP cannot be used instead of substrate GTP, whereas 2'-dGTP and dd-GTP are able to substitute for GTP in the reaction. ITP also can be a substrate as an analog of GTP and as an analog of IMP. Two intermediates of purine nucleotide biosynthesis de novo, 5-amino-4-(N-succinocarboxamide)imidazole ribonucleotide (ASCIR) and 5-amino-4-carbamoyl-imidazole ribonucleotide (ACIR), inhibit AS-synthetase. Hydroxylamine and aspartate analogs also inhibit the enzyme. Effective binding requires a four-carbon-atom chain and unsubstituted amino group; the charge of the beta-carboxy group is not necessary. Comparison of primary structures and substrate specificity of yeast ASCIR- and AS-synthetases suggests independent origin of these proteins.

Adenylosuccinate synthetase from maize. Purification, properties, and mechanism of inhibition by 5'-phosphohydantocidin.

Adenylosuccinate synthetase (AdSS) is the site of action hydantocidin, a potent microbial phytotoxin. A kinetic analysis of the mode of inhibition of a plant adenylosuccinate synthetase by the active metabolite 5'-phosphohydantocidin (5'-PH) was the objective of the present study. AdSS was purified 5800-fold from maize (Zea mays), to our knowledge the first purification of the enzyme from a plant source. N-terminal sequencing established the cleavage site of the previously published deduced sequence of the initial transcript. The subunit molecular mass was determined to be 48 kD and the isoelectric point was at pH 6.1. Values of the Michaelis constant for the three substrates IMP, GTP, and aspartate were 21, 16, and 335 microM, respectively. Inhibition of AdSS by 5'-PH was measurably time-dependent. The trace of the inactivation curve could not be altered by preincubating the enzyme and inhibitor in the absence of substrates but could be linearized by preincubating the enzyme with inhibitor, aspartate, GTP (or GDP), and inorganic phosphate. Inhibition of AdSS by 5'-PH was competitive with IMP, with an apparent Ki of 22 nM. Apparently, 5'-PH inhibits the enzyme by binding to the IMP site and forming a tight, dead-end complex.

Identification of arginine residues in the putative L-aspartate binding site of Escherichia coli adenylosuccinate synthetase.

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.

[Saccharomyces cerevisiae ADE12 gene, coding for adenylosuccinate synthetase (EC 6.3.4.4). Cloning, sequencing, expression, and superproduction]. / Gen ADE12 drozhzhei Saccharomyces cerevisiae, kodiruiushchii adenilosuktsii-sintetazu (KF 6.3.4.4). Klonirovanie, sekvenirovanie, izuchenie ékspressii, superproduktsiia.

The cloning and sequencing of Saccharomyces cerevisiae ADE12 gene encoding the structure of adenylsuccinate synthetase (ASS) are reported for the first time. Comparative analysis of all known ASS sequences was carried out. Regulation of ADE12 gene expression by exogenic adenine was carried out. The properties of ASS which was isolated and purified from overproducing yeast strain were examined.

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.

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.

Characterization of two novel single-stranded DNA-specific autonomously replicating sequence-binding proteins from Saccharomyces cerevisiae, one of which is adenylosuccinate synthetase.

We report here the identification and characterization of two novel proteis from Saccharomyces cerevisiae that bind to the T-rich strand of the core consensus autonomously replicating sequence (ARS) in a highly specific manner. The two proteins, 40 and 45 kDa in size, can be distinguished by multiple criteria from each other and from the 65-kDa ssArS-T-binding protein identified recently in our laboratory (Schmidt, A. M. A., Herterich, S. U., and Krauss, G. (1991) EMBO J. 10, 981-985). The specificity of binding is inferred from gel shift and nuclease-footprinting experiments using single-stranded probes containing the core consensus ARS. With a 321-nucleotide single-stranded ARS1 fragment, specific protection of the A and B1 domain against DNase I digestion is observed. Partial amino acid sequencing and enzymatic assays identify the 45-kDa protein as adenylosuccinate synthetase, an enzyme necessary for the de novo synthesis of adenylate.

Two purine biosynthetic enzymes that are required for cadmium tolerance in Schizosaccharomyces pombe utilize cysteine sulfinate in vitro.

In plants and in certain fungi, exposure to heavy metals induces the synthesis of metal-binding peptides commonly known as phytochelatins. With cadmium, phytochelatins can sequester the metal into a sulfide-containing complex. From genetic analysis of fission yeast mutants, we previously reported that two genes in purine biosynthesis, encoding adenylosuccinate synthetase and succinoaminoimidazole carboxamide ribonucleotide (SAICAR) synthetase, are required for the biogenesis of the phytochelatin-cadmium-sulfide complex in vivo. We suggested that a sulfur analog of aspartate, cysteine sulfinate, might be utilized by these enzymes and that the cysteine sulfinate-derived products would then become intermediates or carriers in a sulfur transfer pathway leading to the sulfide found within the metal chelate. In this paper, we report that partially purified adenylosuccinate synthetase and SAICAR synthetase are capable of utilizing cysteine sulfinate in vitro to form sulfur analog products. Adenylosuccinate lyase, however, fails to catalyze further conversion of these sulfur derivatives. These observations support the genetic data implicating a link among purine biosynthetic enzymes, sulfur metabolism, and cadmium tolerance.

Identification of the purA gene encoding adenylosuccinate synthetase in Thiobacillus ferrooxidans.

The purA gene of Thiobacillus ferrooxidans encoding adenylosuccinate synthetase [EC 6.3.4.4] was identified in the upstream region of the iro gene encoding Fe(II)-oxidase (J. Biol. Chem 267:11242-11247, 1992). The purA gene consisted of 1290 base-pairs, which translated into a 29-amino-acid protein. The gene is functionally active, because it is able to complement an Escherichia coli purA-deficient strain. The deduced gene product has a high degree (60.9%) of sequence identity with that (432 aa) of E. coli purA gene, and both the products share GDEGKGK-DETG-TKLD sequences which are supposed to be GTP-binding domain. The downstream region of the iro gene contained another open-reading frame (ORF) of 1218 bp, and this showed high homology (56.6% over 249 bp) with E. coli ORF-II, which is found as a second ORF and truncated form in the downstream region of the purA gene. Comparison of the gene organization in the flanking region of purA gene between T. ferrooxidans and E. coli is also described.

Molecular cloning and expression of a mouse muscle cDNA encoding adenylosuccinate synthetase.

Adenylosuccinate synthetase (EC 6.3.4.4) catalyzes the first step in formation of AMP from IMP. At least two isozymes exist in vertebrate tissue. An acidic form, present in most tissues, has been suggested to be involved in de novo biosynthesis while a basic isozyme, which predominates in muscle, appears to function in the purine nucleotide cycle. Antibodies specific for the basic isozyme detect a single protein in mouse tissues with highest levels in skeletal muscle, tongue, esophagus, and heart tissue consistent with a role for the enzyme in muscle metabolism. A series of degenerate oligonucleotides were constructed based on peptide sequences from purified rat muscle enzyme and then used to clone a mouse muscle cDNA encoding the basic isozyme. The clone contains a open reading frame of 1356 bases with 452 amino acids. Northern analysis of RNA from mouse tissues showed a tissue distribution similar to that of the protein, indicating a high level of gene expression in muscle. Transfection of COS cells with the mouse muscle cDNA allows expression of a functional protein with a molecular mass of approximately 50 kDa, consistent with the open reading frame and the size of the isolated rat enzyme. The deduced amino acid sequence of the mouse synthetase is 47 and 37% identical to the synthetase sequences from Dictyostelium discoideum and Escherichia coli, respectively. The availability of antibodies and cDNA clones specific for the basic isozyme of adenylosuccinate synthetase from muscle will facilitate future genetic and biochemical analysis of this protein and its role in muscle physiology.

Purification and cDNA-derived sequence of adenylosuccinate synthetase from Dictyostelium discoideum.

Adenylosuccinate synthetase (IMP:L-aspartate ligase (GDP), EC 6.3.4.4) 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.

Overproduction, purification, and characterization of adenylosuccinate synthetase from Escherichia coli.

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.

Nitro analogs of substrates for adenylosuccinate synthetase and adenylosuccinate lyase.

The reactivities of the nitro analogs of the substrates of adenylosuccinate synthetase and adenylosuccinate lyase, the enzymes which catalyze the penultimate and last step, respectively, in the pathway for AMP biosynthesis have been examined. Alanine-3-nitronate, an aspartate analog, was a substrate for the synthetase from Azotobacter vinelandii, having a kcat/Km which was approximately 30% that for aspartate. The product of this reaction was N6-(L-1-carboxy-2-nitroethyl)-AMP. Of nine other substrate analogs tested, only cysteine sulfinate (having 5.5% of the activity of aspartate) was reactive. These results demonstrate the strict requirement of the synthetase for a negatively charged substituent, with a carboxylate-like geometry, at the beta-carbon of the alpha-amino acid substrate. The lyase, purified to homogeneity from brewer's yeast by a new procedure, did not utilize N6-(L-1-carboxy-2-nitroethyl)-AMP as a substrate. However, the nitronate form of this analog was a good inhibitor of the lyase (Km/Ki = 28 when compared to adenylosuccinate), suggesting that it mimics a carbanionic intermediate in the reaction pathway. The avid binding of bromphenol blue by the lyase (Ki = 0.95 microM) was used for active site titrations and for displacement of the enzyme, in the purification protocol, from blue Sepharose.

Effect of acidosis and uninephrectomy on isozymes of adenylosuccinate synthetase in rat kidney.

Normal rat kidney contains primarily the L isozyme of adenylosuccinate synthetase. The increase in total adenylosuccinate synthetase activity that occurs in response to NH4Cl-feeding or a low potassium diet is mainly due to increase in the L isozyme, rather than to an increase in the M isozyme. 1 day after uninephrectomy there is little change in total adenylosuccinate synthetase activity or isozyme distribution in the remaining kidney. These results do not support extension to kidney of the theory proposed for liver that the L isozyme is involved in purine biosynthesis while the M isozyme is involved in ammonia production from amino acids via the purine nucleotide cycle.