Aromatic amino acid aminotransferase of Escherichia coli: nucleotide sequence of the tyrB gene

The tyrB gene of E. coli K-12, which encodes aromatic amino acid aminotransferase (EC 2.6.1.57) was cloned. The nucleotide sequence of about 2 kilobase pairs containing the gene was determined. The coding region of the tyrB gene and the deduced amino acid sequence revealed that the aromatic amino acid aminotransferase of E. coli is homologous with the aspartate aminotransferase.     

Aspartate aminotransferase of Escherichia coli: nucleotide sequence of the aspC gene

The nucleotide sequence of the aspartate aminotransferase [EC 2.6.1.1] structural gene, aspC, of Escherichia coli K-12 was determined. The coding region of the aspC gene contained 1,188 nucleotide residues and encoded 396 amino acid residues. The amino acid sequence deduced from the nucleotide sequence agreed perfectly with that of the protein recently determined for the aspartate aminotransferase of E. coli B (Kondo, K., Wakabayashi, S., Yagi, T., & Kagamiyama, H. (1984) Biochem. Biophys. Res. Commun. 122, 62-67).     

Complete amino acid sequence of rat liver cytosolic alanine aminotransferase

The complete amino acid sequence of rat liver cytosolic alanine aminotransferase (EC 2.6.1.2) is presented. Two primary sets of overlapping fragments were obtained by cleavage of the pyridylethylated protein at methionyl and lysyl bonds with cyanogen bromide and Achromobacter protease I, respectively. The protein was found to be acetylated at the amino terminus and contained 495 amino acid residues. The molecular weight of the subunit was calculated to be 55,018 which was in good agreement with a molecular weight of 55,000 determined by SDS-PAGE and also indicated that the active enzyme with a molecular weight of 114,000 was a homodimer composed of two identical subunits. No highly homologous sequence was found in protein sequence databases except for a 20-residue sequence around the pyridoxal 5'-phosphate binding site of the pig heart enzyme [Tanase, S., Kojima, H., & Morino, Y. (1979) Biochemistry 18, 3002-3007], which was almost identical with that of residues 303-322 of the rat liver enzyme. In spite of rather low homology scores, rat alanine aminotransferase is clearly homologous to those of other aminotransferases from the same species, e.g., cytosolic tyrosine aminotransferase (24.7% identity), cytosolic aspartate aminotransferase (17.0%), and mitochondrial aspartate aminotransferase (16.0%). Most of the crucial amino acid residues hydrogen-bonding to pyridoxal 5'-phosphate identified in aspartate aminotransferase by X-ray crystallography are conserved in alanine aminotransferase. This suggests that the topology of secondary structures characteristic in the large domain of other alpha-aminotransferases with known tertiary structure may also be conserved in alanine aminotransferase.     

Molecular characterization and expression of the gene encoding aspartate aminotransferase from the Pacific oyster Crassostrea gigas exposed to environmental stressors

A partial cDNA encoding cytosolic aspartate aminotransferase (AST) (EC 2.6.1.1) was isolated from a Crassostrea gigas digestive gland library. This sequence was used to design specific primers to amplify the AST genomic sequence. We obtained a complete gene, 5054 bp in length, encoding cytosolic AST and containing a 404 amino acid open reading frame. Phylogenetic analysis showed that C. gigas AST sequence constitutes a branch distinct from homologous sequences from other invertebrate groups. We also investigated AST mRNA expression in different tissues of oysters exposed to hydrocarbons, pesticides, hypoxia and hypo-salinity stress. The results showed that AST expression responds to hydrocarbon exposure, hypoxia and salinity stress, but not to pesticide exposure in an organ and time-specific manner. Use of AST as a potential molecular biomarker for monitoring of disturbed ecosystems is discussed.     

Glutamine:phenylpyruvate aminotransferase from an extremely thermophilic bacterium, Thermus thermophilus HB8

A subfamily I aminotransferase gene homologue containing an open reading frame encoding 381 amino acid residues (Mr=42,271) has been identified in the process of the genome project of an extremely thermophilic bacterium, Thermus thermophilus HB8. Alignment of the predicted amino acid sequence using FASTA shows that this protein is a member of aminotransferase subfamily Igamma. The protein shows around 40% identity with both T. thermophilus aspartate aminotransferase [EC 2.6.1.1] and mammalian glutamine:phenylpyruvate aminotransferase [EC 2.6.1.64]. The recombinant protein expressed in Escherichia coli is a homodimer with a subunit molecular weight of 42,000, has one pyridoxal 5'-phosphate per subunit, and is highly active toward glutamine, methionine, aromatic amino acids, and corresponding keto acids, but has no preference for alanine and dicarboxylic amino acids. These substrate specificities are similar to those described for mammalian glutamine: phenylpyruvate aminotransferase. This is the first enzyme reported so far that has the glutamine aminotransferase activity in non-eukaryotic cells. As the presence of aromatic amino acid:2-oxoglutarate aminotransferase [EC 2.6.1.57] has not been reported in T. thermophilus, this enzyme is expected to catalyze the last transamination step of phenylalanine and tyrosine biosynthesis. It may also be involved in the methionine regeneration pathway associated with polyamine biosynthesis. The enzyme shows a strikingly high pKa value (9.3) of the coenzyme Schiff base in comparison with other subfamily I aminotransferases. The origin of this unique pKa value and the substrate specificity is discussed based on the previous crystallographic data of T. thermophilus and E. coli aspartate aminotransferases.     

Distribution of two aminotransferases and D-amino acid oxidase within the nephron of young and adult rats.

In the adult rat kidney, alanine aminotransferase (EC 2.6.1.2), aspartate aminotransferase (EC 2.6.1.1) and D-amino acid oxidase (EC 1.4.3.3) were measured in glomeruli, 4 parts of the proximal tubule, 2 parts of the distal tubule and in patches from the thin limb area and the papilla. These enzymes were measured in more limited parts of the nephron during postnatal development. Adult aspartate aminotransferase activities (percentage of the highest) ranged from 100 in the distal straight segment to 25 in the late part of the proximal straight segment to 10 in the thin limb and papillary area. Alanine aminotransferase (lower by a factor of 100 in absolute terms) was distributed as the mirror image of aspartate aminotransferase within proximal and distal tubules. D-Amino acid oxidase was 850-fold higher in proximal straight segments than in medullary structures. During development alanine aminotransferase increased 6-fold and D-amino acid oxidase, 4.5-fold in proximal straight tubules but aspartate aminotransferase increased in distal straight tubles 8-fold.     

Rat cytosolic aspartate aminotransferase: molecular cloning of cDNA and expression in Escherichia coli

cDNA clones for rat cytosolic aspartate aminotransferase (cAspAT, L-aspartate:2-oxoglutarate aminotransferase) [EC 2.6.1.1] were isolated from a rat cDNA library, and the primary structure of the gene for cAspAT was deduced from its cDNA sequence. Rat cAspAT consists of 412 amino acids and its molecular weight is 46,295. The deduced amino acid sequence of rat cAspAT was compared with the sequences of AspATs from other species. The degree of sequence identities of rat/mouse cAspAT, rat/pig cAspAT, rat/chicken cAspAT, rat/pig mAspAT, and rat/Escherichia coli AspAT were 97.1, 89.6, 81.7, 48.1, and 41.2%, respectively. A coding region of rat cAspAT cDNA was inserted into E. coli expression vector pUC9, and enzymatically active cAspAT was expressed as a beta-galactosidase-cAspAT hybrid protein. This hybrid protein represented about 18% of the soluble proteins in E. coli and its kinetic properties were comparable with those of cAspAT preparations purified from rat liver.     

The primary structure of mitochondrial aspartate aminotransferase from human heart

The complete amino acid sequence of the mitochondrial aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) from human heart has been determined based mainly on analysis of peptides obtained by digestion with trypsin and by chemical cleavage with cyanogen bromide. Comparison of the sequence with those of the isotopic isoenzymes from pig, rat and chicken showed 27, 29 and 55 differences, respectively, out of a total of 401 amino acid residues. Evidence for structural microheterogeneity at position 317 has also been obtained.     

Activities of enzymes concerned with pyruvate and oxaloacetate metabolism in the heart and liver of developing sheep.

1. In order to assess whether the potential ability of heart ventricular muscle and liver to metabolise substrates such as alanine, aspartate and lactate varies as the sheep matures and its nutrition changes, the activities of the following enzymes were determined in tissues of lambs obtained at varying intervals between 50 days after conception to 16 weeks after birth and in livers from adult pregnant ewes: lactate dehydrogenase (EC 1.1.1.27), alanine aminotransferase (EC 2.6.1.2), pyruvate kinase (EC 2.7.1.40), pyruvate carboxylase (EC 6.4.1.1), phosphoenolpyruvate carboxykinase (GTP)(EC 4.1.1.32), malate dehydrogenase (EC 1.1.1.37), aspartate aminotransferase (EC 2.6.1.1) and citrate (si)-synthase (EC 4.1.3.7). 2. In the heart a most marked increase in alanine aminotransferase activity was found throughout development. During this period the activities of citrate (si)-synthase, lactate dehydrogenase and pyruvate carboxylase also increased. There were no substantial changes in the activities of aspartate aminotransferase, malate dehydrogenase or pyruvate kinase. Pyruvate kinase activities were five times greater in the heart compared with those found in the liver. No significant activity of phosphoenolpyruvate carboxykinase (GTP) was detected in heart muscle. 3. In the liver the activities of both alanine aminotransferase and aspartate aminotransferase increased immediately following birth although the activity of alanine aminotransferase was lower in livers of pregnant ewes than in any of the lambs. As with alanine aminotransferase the highest activities of lactate dehydrogenase were found during the period of postnatal growth. No marked changes were observed in malate dehydrogenase or citrate (si)-synthase activities during development. A small decline in pyruvate kinase activity occurred whilst the activities of pyruvate carboxylase and phosphoenolpyruvate carboxykinase (GTP) tended to rise during development.     

Similarity between pyridoxal/pyridoxamine phosphate-dependent enzymes involved in dideoxy and deoxyaminosugar biosynthesis and other pyridoxal phosphate enzymes.

A multiple sequence alignment among aspartate aminotransferase, dialkylglycine decarboxylase, and serine hydroxymethyltransferase (DAS) was used for profile databank search. The DAS profile could detect similarities to other pyridoxal or pyridoxamine phosphate-dependent enzymes, like several gene products involved in dideoxysugar and deoxyaminosugar synthesis. The alignment among DAS and such gene products shows the conservation of aspartate 222 and lysine 258, which, in aspartate aminotransferase, interacts with the N1 of the coenzyme pyridine ring and forms the internal Schiff base, respectively. The lysine is replaced by histidine in the pyridoxamine phosphate-dependent gene products. The alignment indicates also that the region encompassing the coenzyme binding site is the most conserved.     

CEREBRAL AROMATIC AMINOTRANSFERASE

—Aromatic: 2-oxoglutarate aminotransferase has been purified about 950-fold from rat brain mitochondria. The purified enzyme was homogeneous in polyacrylamide gel electrophoresis and had a molecular weight of approx 63,000. On the basis of substrate specificity, substrate inhibition, purification ratio, yield, polyacrylamide gel electrophoresis and some other properties of the enzyme it has been suggested that brain mitochondrial tyrosine:2-oxoglutarate aminotransferase (l -tyrosine: 2-oxoglutarate aminotransferase, EC 2.6.1.5) is identical with brain mitochondrial phenylalanine and kynurenine: 2-oxoglutarate aminotransferases (l -kynurenine: 2-oxoglutarate aminotransferase, EC 2.6.1.7), and also with aspartate: 2-oxoglutarate aminotransferase (l -aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1).     

Effect of oxidized glutathione on some enzymes of erythrocytes and its relation to erythrocytic enzyme activity and electrophoretic mobility

1. Oxidized glutathione reacts or interacts with some erythrocytic enzymes (glucose 6-phosphate dehydrogenase, EC 1.1.1.49, aspartate aminotransferase, EC 2.6.1.10) but not with some others (lactate dehydrogenase, EC 1.1.1.27). 2. GSSG does not diminish the activity of any of these enzymes and is therefore not responsible for the decreased enzyme activities associated with older erythrocytes. 3. It may be that the reaction of aspartate aminotransferase with GSSG is the cause for the more rapid anodic electrophoretic mobility of this enzyme derived from human erythrocytes when compared with the mobility of the same enzyme from other human tissues. 4. A reinterpretation of some related, previously published, data with regard to the electrophoretic mobility of the above-mentioned enzymes from young and old erythrocytes is presented.     

Developmental change of endogenous glutamate and gamma-glutamyl transferase in cultured cerebral cortical interneurons and cerebellar granule cells,and in mouse cerebral cortex and cerebellum in vivo

The developmental change of endogenous glutamate, as correlated to that of gamma-glutamyl transferase and other glutamate metabolizing enzymes such as phosphate activated glutaminase, glutamate dehydrogenase and aspartate, GABA and ornithine aminotransferases, has been investigated in cultured cerebral cortex interneurons and cerebellar granule cells. These cells are considered to be GABAergic and glutamatergic, respectively. Similar studies have also been performed in cerebral cortex and cerebellum in vivo. The developmental profiles of endogenous glutamate in cultured cerebral cortex interneurons and cerebellar granule cells corresponded rather closely with that of gamma-glutamyl transferase and not with other glutamate metabolizing enzymes. In cerebral cortex and cerebellum in vivo the developmental profiles of endogenous glutamate, gamma-glutamyl transferase and phosphate activated glutaminase corresponded with each other during the first 14 days in cerebellum, but this correspondence was less good in cerebral cortex. During the time period from 14 to 28 days post partum the endogenous glutamate concentration showed no close correspondence with any particular enzyme. It is suggested that gamma-glutamyltransferase regulates the endogenous glutamate concentration in culture neurons. The enzyme may also be important for regulation of endogenous glutamate in brain in vivo and particularly in cerebellum during the first 14 days post partum. Gamma-glutamyl transferase in cultured neurons and brain tissue in vivo appears to be devoid of maleate activated glutaminase.Abbreviations used Asp-T aspartate aminotransferase (EC 2.6.1.1) - GABA-T GABA aminotransferase (EC 2.6.1.19) - GAD glutamate decarboxylase (EC 4.1.1.15) - gamma-GT gamma-glutamyl transferase (gamma-glutamyl transpeptidase) (EC. 2.3.2.2) - Glu glutamate - GDH glutamate dehydrogenase (EC 1.4.1.3) - GS glutamine synthetase (EC 6.3.1.2) - MAG maleate activated glutaminase - Orn-T ornithine aminotransferase (EC 2.6.1.13) - PAG phosphate activated glutaminase (EC 3.5.1.1)     

The cloning and sequence analysis of the aspC and tyrB genes from Escherichia coli K12. Comparison of the primary structures of the aspartate aminotransferase and aromatic aminotransferase of E. coli with those of the pig aspartate aminotransferase isoenzymes.

In this paper we describe the cloning and sequence analysis of the tyrB and aspC genes from Escherichia coli K12, which encode the aromatic aminotransferase and aspartate aminotransferase respectively. The tyrB gene was isolated from a cosmid carrying the nearby dnaB gene, identified by its ability to complement a dnaB lesion. Deletion and linker insertion analysis located the tyrB gene to a 1.7-kilobase NruI-HindIII-digest fragment. Sequence analysis revealed a gene encoding a 43 000 Da polypeptide. The gene starts with a GTG codon and is closely followed by a structure resembling a rho independent terminator. The aspC gene was cloned by screening gene banks, prepared from a prototrophic E. coli K12 strain, for plasmids able to complement the aspC tyrB lesions in the aminotransferase-deficient strain HW225. Sub-cloning and deletion analysis located the aspC gene on a 1.8-kilobase HincII-StuI-digest fragment. Sequence analysis revealed the presence of a gene encoding a 43 000 Da protein, the sequence of which is identical with that previously obtained for the aspartate aminotransferase from E. coli B. Considerable overproduction of the two enzymes was demonstrated. We compared the deduced protein sequences with those of the pig mitochondrial and cytoplasmic aspartate aminotransferases. From the extensive homology observed we are able to propose that the two E. coli enzymes possess subunit structures, subunit interactions and coenzyme-binding and substrate-binding sites that are very similar both to each other and to those of the mammalian enzymes and therefore must also have very similar catalytic mechanisms. Comparison of the aspC and tyrB gene sequences reveals that they appear to have diverged as much as is possible within the constraints of functionality and codon usage.     

Investigations of the anaerobic metabolism of the polychaete wormNereis diversicolor M.

Summary The anaerobic metabolism ofNereis diversicolor M. was studied during various periods of experimental anaerobiosis.The degradation of glycogen is shown to be the main source of anaerobic energy production. During first hours of anaerobiosis, aspartate, in addition to glycogen, is metabolized in considerable quantities.Five acids were found to accumulate as end-products: alanine, D-lactate, succinate, acetate and propionate (Table 2).Alanine is accumulated only during the first hours of anaerobiosis. The increase in alanine is correlated with a decrease in aspartate.D-Lactate is the main end-product during the first 24 h of anaerobiosis, and continues to be produced even during prolonged anaerobiosis. In accordance with lactate production,Nereis diversicolor possesses a high glycolytic capacity (Table 4).The major end-products of long term fermentation are propionate and acetate. In contrast to other end-products, these acids are excreted in substantial amounts.Abbreviations GAPDH glyceraldehydephosphate dehydrogenase, EC 1.2.1.12 - LDH lactate dehydrogenase, EC 1.1.1.27 - GOT aspartate aminotransferase, EC 2.6.1.1 - GPT alanine aminotransferase, EC 2.6.1.2 - MDH malate dehydrogenase, EC 1.1.1.37 Supported by Deutsche Forschungsgemeinschaft (Gr 456/5 and Gr 456/6)     

Induction of cytosolic aspartate aminotransferase by glucagon in primary cultured rat hepatocytes

The activity and the mRNA content of cytosolic aspartate aminotransferase (EC 2.6.1.1) were examined in cultured rat hepatocytes. Addition of glucagon (1 x 10(-7) M) in the presence of dexamethasone (1 x 10(-7) M) caused about 2-fold increase in the activity and mRNA content. Dibutyryl cAMP (1 x 10(-4) M) could replace glucagon for this effect. Maximal induction of cytosolic aspartate aminotransferase mRNA was observed 8 h after their additions. Insulin (1 x 10(-7) M) did not inhibit the enzyme induction by glucagon or dibutyryl cAMP. These results suggest that the cytosolic aspartate aminotransferase gene is regulated by cAMP, and not by insulin.     

The sequences of the coenzyme-binding peptide in the cytoplasmic and the mitochondrial aspartate aminotransferases from sheep liver.

The sequences of the coenzyme-binding peptide of both cytoplasmic and mitochondrial aspartate aminotransferases from sheep liver were determined. The holoenzymes were treated with NaBH4 and digested with chymotrypsin; peptides containing bound pyridoxal phosphate were then isolated. One phosphopyridoxyl peptide was obtained from sheep liver cytoplasmic aspartate aminotransferase. Its sequence was Ser-Ne-(phosphopyridoxyl)-Lys-Asn-Phe. This sequence is identical with that reported for the homologous peptide from pig heart cytoplasmic aspartate aminotransferase. Two phosphopyridoxyl peptides with different RF values were isolated from the sheep liver mitochondrial isoenzyme. They had the same N-terminal amino acid and similar amino acid composition. The mitochondrial phosphopyridoxyl peptide of highest yield and purity had the sequence Ala-Ne-(phosphopyridoxyl)-Lys-Asx-Met-Gly-Leu-Tyr. The sequence of the first four amino acids is identical with that already reported for the phosphopyridoxyl tetrapeptide from the pig heart mitochondrial isoenzyme. The heptapeptide found for the sheep liver mitochondrial isoenzyme closely resembles the corresponding sequence taken from the primary structure of the pig heart cytoplasmic aspartate aminotransferase.