Crystal structure of Saccharomyces cerevisiae cytosolic aspartate aminotransferase.

The crystal structure of Saccharomyces cerevisiae cytoplasmic aspartate aminotransferase (EC 2.6.1.1) has been determined to 2.05 A resolution in the presence of the cofactor pyridoxal-5'-phosphate and the competitive inhibitor maleate. The structure was solved by the method of molecular replacement. The final value of the crystallographic R-factor after refinement was 23.1% with good geometry of the final model. The yeast cytoplasmic enzyme is a homodimer with two identical active sites containing residues from each subunit. It is found in the "closed" conformation with a bound maleate inhibitor in each active site. It shares the same three-dimensional fold and active site residues as the aspartate aminotransferases from Escherichia coli, chicken cytoplasm, and chicken mitochondria, although it shares less than 50% sequence identity with any of them. The availability of four similar enzyme structures from distant regions of the evolutionary tree provides a measure of tolerated changes that can arise during millions of years of evolution.     

The three-dimensional structure of mitochondrial aspartate aminotransferase at 4.5 A resolution

An X-ray crystallographic study at 4.5 Å resolution has been carried out with triclinic crystals of chicken mitochondrial aspartate aminotransferase.In the electron density map, the enzyme is clearly visible as an isologous α₂-dimer (105 Å × 60 Å × 50 Å) in which the subunits are associated about a molecular 2-fold axis. Each subunit of dimensions 70 Å × 50 Å × 40 Å contains at least seven helices, one of which is about 50 Å long.Difference maps have revealed the positions of the pyridoxyl and the phosphate moieties of the coenzyme as well as the general substrate binding area. The active sites are on opposite sides of the dimer, about 30 Å apart and close to the intersubunit boundary, so that probably both subunits contribute to each active site. An isolated chain segment, passing in front of the active site and ending in contact with the neighbouring subunit is interpreted as one of the chain termini.     

Thermal inactivation, denaturation and aggregation of mitochondrial aspartate aminotransferase

A comparative study of thermal denaturation and inactivation of aspartate aminotransferase from pig heart mitochondria (mAAT) has been carried out (10 mM Na phosphate buffer, pH 7.5). Analysis of the data on differential scanning calorimetry shows that thermal denaturation of mAAT follows the kinetics of irreversible reaction of the first order. The kinetics of thermal inactivation of mAAT follows the exponential law. It has been shown that the inactivation rate constant (k_in) is higher than the denaturation rate constant (k_den). The k_in/k_den ratio decreases from 28.8 ± 0.1 to 1.30 ± 0.09 as the temperature increases from 57.5 to 77 °C. The kinetic model explaining the discrepancy between the inactivation and denaturation rates has been proposed. The size of the protein aggregates formed at heating of mAAT at a constant rate (1 °C min^{− 1}) has been characterized by dynamic light scattering.     

Glycation-induced inactivation of aspartate aminotransferase, effect of uric acid

Glycation is common posttranslational modification of proteins impairing their function, which occurs during diabetes mellitus and aging. Beside extracellular glycation of long-lived proteins, intracellular modifications of short-lived proteins by more reactive sugars like fructose are possible. The process includes free oxygen radicals (glycoxidation). In an attempt to reduce glycoxidation and formation of advanced glycation products (AGE), influence of 0.2–1.2 mM uric acid as endogenous antioxidant on glycoxidation of purified pig heart aspartate aminotransferase (AST) by 50 mM and 500 mM D-fructose in vitro was studied. Uric acid at 1.2 mM concentration reduced AST activity decrease and formation of total AGE products caused by incubation in vitro of the enzyme with sugar up to 25 days at 37 °C. The results thus support the hypothesis that uric acid has beneficial effects in controlling protein glycoxidation. The in vitro system AST-fructose proved to be a useful tool for investigation of glycation process. (Mol Cell Biochem 278: 85–92, 2005)     

Redesign of the substrate specificity of Escherichia coli aspartate aminotransferase to that of Escherichia coli tyrosine aminotransferase by homology modeling and site-directed mutagenesis.

Although several high-resolution X-ray crystallographic structures have been determined for Escherichia coli aspartate aminotransferase (eAATase), efforts to crystallize E. coli tyrosine aminotransferase (eTATase) have been unsuccessful. Sequence alignment analyses of eTATase and eAATase show 43% sequence identity and 72% sequence similarity, allowing for conservative substitutions. The high similarity of the two sequences indicates that both enzymes must have similar secondary and tertiary structures. Six active site residues of eAATase were targeted by homology modeling as being important for aromatic amino acid reactivity with eTATase. Two of these positions (Thr 109 and Asn 297) are invariant in all known aspartate aminotransferase enzymes, but differ in eTATase (Ser 109 and Ser 297). The other four positions (Val 39, Lys 41, Thr 47, and Asn 69) line the active site pocket of eAATase and are replaced by amino acids with more hydrophobic side chains in eTATase (Leu 39, Tyr 41, Ile 47, and Leu 69). These six positions in eAATase were mutated by site-directed mutagenesis to the corresponding amino acids found in eTATase in an attempt to redesign the substrate specificity of eAATase to that of eTATase. Five combinations of the individual mutations were obtained from mutagenesis reactions. The redesigned eAATase mutant containing all six mutations (Hex) displays second-order rate constants for the transamination of aspartate and phenylalanine that are within an order of magnitude of those observed for eTATase. Thus, the reactivity of eAATase with phenylalanine was increased by over three orders of magnitude without sacrificing the high transamination activity with aspartate observed for both enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular cloning of a cDNA encoding aspartate aminotransferase-P2 from lupin root nodules

Two isoenzymic forms of aspartate aminotransferase are present in the plant fraction of developing lupin root nodules. One of these forms, aspartate aminotransferase-P₂ (AAT-P₂), increases dramatically with the onset of biological nitrogen fixation and is associated with the assimilation of ammonia by the plant in the Rhizobium-legume symbiosis. A day 18 lupin nodule cDNA library in the ZapII vector was immunoscreened with a monoclonal antibody specific for AAT-P₂ and yielded two near-full-length 1700 bp clones. These clones were sequenced. Amino acid sequences from three peptides derived from immunopurified AAT-P₂ were aligned, and showed 100% homology with the amino acid sequence deduced from the cDNA clones. The DNA sequence showed 50% homology with AAT sequences from a range of animal sources. Conversion of the clones to the phagemid form allowed their expression in Escherichia coli where both exhibited enzyme activity that could be immunoprecipitated with AAT-P₂-specific monoclonal antibodies. Western blot analysis revealed protein moieties with molecular masses of 39, 43, 45 and 55 kDa. The 5 end of the clones coded for a hydrophobic leader sequence of about 50 amino acids indicative of a targeting sequence and consistent with the plastid localisation of nodule AAT-P₂.     

Equilibrium kinetics of substrate-enzyme interactions in single crystals of cytoplasmic aspartate aminotransferase

Orthorhombic single crystals of cytoplasmic aspartate aminotransferase were examined alone or in the presence of substrates or inhibitors to quantitatively compare the interaction of ligands with the active-site chromophore between soluble and crystalline enzyme. As in enzyme solutions, equilibrium kinetic measurements can be made between substrates and single crystals of cytoplasmic aspartate aminotransferase. The absorption spectra of ligand-free enzyme forms and of enzyme-substrate or-inhibitor complexes are as distinctive as when the enzyme is in solution. The dissociation constants for glutamate with the pyridoxal form of the enzyme are identical to those in solution. The substrate analog erythro--hydroxyaspartate also binds with equal affinity to the active site in enzyme crystals as in solution; and the affinity of -ketoglutarate to bind in nonproductive complexes with the pyridoxal form of the enzyme is also unimpaired in the crystal (K _d =2 mM). In contrast to the affinity constants, the stoichiometry of the interactions does not appear to correlate to those in solution. In the presence of an amino acid plus keto acid substrates pair, the absorbance values of the enzyme-substrate complex(es) could be interpreted as for occupany of only half the available sites in the crystals. Yet an amino acid, cysteine sulfinate, and -keto acids such as , -difluorooxalacetate convert all active sites in the crystal to the pyridoxamine or pyridoxal form when added to the pyridoxal or pyridoxamine forms, respectively. This ability to completely undergo substrate-induced half-transamination and the apparently conflicting results in trapping half the sites in enzyme-substrate complexes are incorporated into a proposed reciprocating mechanism applicable only to the crystalline state of the enzyme and dictated by crystal packing forces rather than an intrinsic property of the enzyme. Active-site bound pyridoxal phosphate continues to behave as a pH indicator; nevertheless, the pK value of the single crystals is a pH unit (pK=7.15) higher than that in solution. This variation is interpreted as indication of a difference in the environment of the chromophore between the crystal and solution states. While the environmental difference does not significantly alter the affinity for substrates, it could account for the reduced rates in transformation of the enzyme-substrate complexes in half-transamination reactions in the crystalline state.     

Characterization of a soybean cDNA clone encoding the mitochondrial isozyme of aspartate aminotransferase,AAT4

A soybean leaf cDNA clone, pSAT2, was isolated by hybridization to a carrot aspartate aminotransferase (EC 2.6.1.1.; AAT) cDNA clone at low stringency. pSAT2 contained an open reading frame encoding a 47640 Da protein. The protein encoded by pSAT2 showed significant sequence similarity to AAT proteins from both plants and animals. It was most similar to two Panicum mitochondrial AATs, 81.5% and 82.0% identity. Alignment of the pSAT2-encoded protein with other mature AAT enzymes revealed a 25 amino acid N-terminal extension with characteristics of a mitochondrial transit peptide. A plasmid, pEXAT2, was constructed to encode the mature pSAT2 protein lacking the putative mitochondrial transit peptide. Escherichia coli containing the plasmid expressed a functional AAT isozyme which comigrated with the soybean AAT4 isozyme during agarose gel electrophoresis. Equilibrium sucrose gradient sedimentation of soybean extracts demonstrated that AAT4 specifically cofractionated with mitochondria. Antibodies raised against the pEXAT2-encoded AAT protein reacted with AAT4 of soybean and not with other AAT isozymes detected in soybean tissues, providing further evidence that clone pSAT2 encodes the soybean mitochondrial isozyme AAT4.     

Generation process of cytosolic aspartate aminotransferase molecular forms by several treatments

-, -, and -forms of chicken liver cytosolic aspartate aminotransferase generate variants on storage (4°C, 25 days). The variants developed from each isolated form appeared as evenly spaced bands with increasing anodic mobilities after polyacrylamide gel electrophoresis (PAGE), pH 8.8, and specific staining. Their mobilities coincided with those of the more negatively charged forms present in fresh tissue. Development of faster-running variants on storage was avoided by addition of thiol reagents to the freshly isolated forms. In their presence, - and -forms were partially transformed into one and two variants with lower anodic mobilities analogous to those of native - and -forms. Short pH and heat treatments did not modify the electrophoretic patterns of the -, -, and -forms, but the incubation with 5 mMl-ascorbic acid (37C, 7h) produced more anodic active bands. The formation of these variants was inhibited by the presence, in the incubation mixture, of superoxide dismutase and catalase. The kinetic parameters of the forms submitted to the different treatments were similar to those of the freshly isolated subforms. The results obtained suggest that minor subforms of the enzyme could be generatedin vivo by a mechanism in which the oxidation of particular amino acid groups is involved.     

In vitro synthesis of precursor forms of pig heart aspartate aminotransferase isozymes

Precursor forms of the isozymes of aspartate aminotransferase from pig heart were synthesized in vitro and purified by binding to specific antibodies. Analysis by sodium dodecylsulfate polyacrylamide gel electrophoresis showed that the precursor of the cytosolic enzyme has a similar molecular weight to that of the mature protein whereas the precursor of the mitochondrial isozyme has a molecular weight greater than that of the corresponding mature protein (ΔMW ? 2500). Preliminary sequence studies seem to suggest that the precursor of the mitochondrial isozyme has an extra N-terminal peptide sequence while that of the cytosolic protein has only an extra N-terminal methionine residue.     

Yeast aspartate aminotransferase: preparation of the apoenzyme and its interaction with coenzyme analogues

Affinity chromatography of yeast aspartate aminotransferase [l-aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1] on N′(ω-aminohexyl) pyridoxamine-5-phosphate Sepharose 4B is reported. The specific activity of the enzyme obtained, fully activated with pyridoxal-5-phosphate, was higher than that of previous preparations but the yield of purified enzyme was poor. Purification using DEAE-cellulose gave a higher yield of enzyme with lower specific activity. This preparation contained an appreciable amount of the holoenzyme. Use of sodium borohydride permitted the preparation of apoenzyme containing only 1.4% of the holo-form. Four coenzyme analogues were synthesized. These were the N′-acetyl-, the N′-methyl- and the N′-benzyloxycarbonylglycyl-pyridoxamine-5-phosphate and the O-acetylpyridoxal-5-phosphate. The three N′-substituted pyridoxamine-5-phosphate derivatives were all effective inhibitors of the enzyme, while the O-acetylpyridoxal-5-phosphate bound to the apoenzyme and gave an active enzyme.     

Isolation and characterization of a soybean cDNA clone encoding the plastid form of aspartate aminotransferase

Five aspartate aminotransferase (EC 2.6.1.1; AAT) isozymes were identified in soybean seedling extracts and designated AAT1 to AAT5 based on their rate of migration on non-denaturing electrophoretic gels. AAT1 was detected only in extracts of cotyledons from dark-grown seedlings. AAT3 and AAT4 were detected in crude extracts of leaves and in cotyledons of seedlings grown in the light. AAT2 and AAT5 were detected in all tissues examined. A soybean leaf cDNA clone, pSAT17, was identified by hybridization to a carrot AAT cDNA clone at low stringency. pSAT17 had an open reading frame which could encode a 50 581 Da protein. Alignment of the deduced amino acid sequence from the pSAT17 open reading frame with mature AAT protein sequences from rat disclosed a 60 amino acid N-terminal extension in the pSAT17 protein. This extension had characteristics of a plastid transit peptide.A plasmid, pEXAT17, was constructed which encoded the mature protein lacking the putative chloroplast transit polypeptide. Transformed Escherichia coli expressed a functional soybean AAT isozyme, which comigrated with the soybean AAT5 isozyme during agarose gel electrophoresis. Differential sucrose gradient sedimentation of soybean extracts indicated that AAT5 specifically cofractionated with chloroplasts. Antibodies raised against the pEXAT17-encoded AAT protein specifically reacted with the AAT5 isozyme of soybean and not with any of the other isozymes, indicating that the soybean cDNA clone, pSAT17, encodes the chloroplast isozyme, AAT5.     

Simultaneous purification by affinity chromatography of rat liver mitochondrial aspartate aminotransferase and malate dehydrogenase and electrophoretic properties

Mitochondrial aspartate aminotransferase and malate dehydrogenase were purified to homogeneity from rat liver by the use of aspartate-coupled Sepharose, ion exchange, and Blue Sepharose chromatography. This procedure permits rapid preparation of these enzymes. The pI of each enzyme was determined and anomalous electrophoretic properties of aspartate aminotransferase were described.     

Specific inhibition of the aspartate aminotransferase of Plasmodium falciparum

Aspartate aminotransferases (AspATs; EC 2.6.1.1) catalyze the conversion of aspartate and α-ketoglutarate into oxaloacetate and glutamate and are key enzymes in the nitrogen metabolism of all organisms. Recent findings suggest that the plasmodial enzyme [Plasmodium falciparum aspartate aminotransferase (PfAspAT)] may also play a pivotal role in energy metabolism and in the de novo biosynthesis of pyrimidines. However, while PfAspAT is a potential drug target, the high homology between the active sites of currently available AspAT structures hinders the development of specific inhibitors of these enzymes. In this article, we report the X-ray structure of the PfAspAT homodimer at a resolution of 2.8 Å. While the overall fold is similar to the currently available structures of other AspATs, the structure presented shows a significant divergence in the conformation of the N-terminal residues. Deletion of these divergent PfAspAT N-terminal residues results in a loss of activity for the recombinant protein, and addition of a peptide containing these 13 N-terminal residues results in inhibition both in vitro and in a lysate isolated from cultured parasites, while the activity of human cytosolic AspAT is unaffected. The finding that the divergent N-terminal amino acids of PfAspAT play a role in catalytic activity indicates that specific inhibition of the enzyme may provide a lead for the development of novel compounds in the treatment of malaria. We also report on the localization of PfAspAT to the parasite cytosol and discuss the implications of the role of PfAspAT in the supply of malate to the parasite mitochondria.     

Specific features of expression of aspartate aminotransferase genes in early development of some cyprinid fishes and their intergeneric F1 hybrids

Temporal parameters of expression of the aspartate aminotransferase gene Aat-1 parental alleles were studied in early development of intergeneric reciprocal F1 hybrids of the bream, roach, and blue bream. When the first Aat-1 expression was timed to the early stages (late blastula-gastrula), the gene parental alleles were activated asynchronously according to the maternal types (blue bream × roach hybrids). When the first Aat-1 expression was timed to later stages (yolk sac resorption), the parental alleles were activated synchronously (bream × roach, roach × bream, and roach × blue bream hybrids). The pattern of activation of embryonic genes is determined by the maternal environment and the influence of allele interactions is not excluded: Aatf/Aat-sl (bream × roach, roach × bream, and roach × blue bream) and Aat-sl/Aat-med (blue bream × roach).     

Identification and functional analysis of a prokaryotic-type aspartate aminotransferase: implications for plant amino acid metabolism

In this paper, we report the identification of genes from pine (PpAAT), Arabidopsis (AtAAT) and rice (OsAAT) encoding a novel class of aspartate aminotransferase (AAT, EC 2.6.1.1) in plants. The enzyme is unrelated to other eukaryotic AATs from plants and animals but similar to bacterial enzymes. Phylogenetic analysis indicates that this prokaryotic-type AAT is closely related to cyanobacterial enzymes, suggesting it might have an endosymbiotic origin. Interestingly, most of the essential residues involved in the interaction with the substrate and the attachment of pyridoxal phosphate cofactor in the active site of the enzyme were conserved in the deduced polypeptide. The polypeptide is processed in planta to a mature subunit of 45 kDa that is immunologically distinct from the cytosolic, mitochondrial and chloroplastic isoforms of AAT previously characterized in plants. Functional expression of PpAAT sequences in Escherichia coli showed that the processed precursor is assembled into a catalytically active homodimeric holoenzyme that is strictly specific for aspartate. These atypical genes are predominantly expressed in green tissues of pine, Arabidopsis and rice, suggesting a key role of this AAT in nitrogen metabolism associated with photosynthetic activity. Moreover, immunological analyses revealed that the plant prokaryotic-type AAT is a nuclear-encoded chloroplast protein. This implies that two plastidic AAT co-exist in plants: a eukaryotic type previously characterized and the prokaryotic type described here. The respective roles of these two enzymes in plant amino acid metabolism are discussed.