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PubMed2003

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.

Amino Acid Sequence↗

PubMed1995

Imidazole acetol phosphate aminotransferase in Zymomonas mobilis: molecular genetic, biochemical, and evolutionary analyses.

hisH encodes imidazole acetol phosphate (IAP) aminotransferase in Zymomonas mobilis and is located immediately upstream of tyrC, a gene which codes for cyclohexadienyl dehydrogenase. A plasmid containing hisH was able to complement an Escherichia coli histidine auxotroph which lacked the homologous aminotransferase. DNA sequencing of hisH revealed an open reading frame of 1,110 bp, encoding a protein of 40,631 Da. The cloned hisH product was purified from E. coli and estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to have a molecular mass of 40,000 Da. Since the native enzyme had a molecular mass of 85,000 Da as determined by gel filtration, the active enzyme species must be a homodimer. The purified enzyme was able to transaminate aromatic amino acids and histidine in addition to histidinol phosphate. The existence of a single protein having broad substrate specificity was consistent with the constant ratio of activities obtained with different substrates following a variety of physical treatments (such as freeze-thaw, temperature inactivation, and manipulation of pyridoxal 5'-phosphate content). The purified enzyme did not require addition of pyridoxal 5'-phosphate, but dependence upon this cofactor was demonstrated following resolution of the enzyme and cofactor by hydroxylamine treatment. Kinetic data showed the classic ping-pong mechanism expected for aminotransferases. Km values of 0.17, 3.39, and 43.48 mM for histidinol phosphate, tyrosine, and phenylalanine were obtained. The gene structure around hisH-tyrC suggested an operon organization. The hisH-tyrC cluster in Z. mobilis is reminiscent of the hisH-tyrA component of a complex operon in Bacillus subtilis, which includes the tryptophan operon and aroE. Multiple alignment of all aminotransferase sequences available in the database showed that within the class I superfamily of aminotransferases, IAP aminotransferases (family I beta) are closer to the I gamma family (e.g., rat tyrosine aminotransferase) than to the I alpha family (e.g., rat aspartate aminotransferase or E. coli AspC). Signature motifs which distinguish the IAP aminotransferase family were identified in the region of the active-site lysine and in the region of the interdomain interface.

Amino Acid Sequence↗

PubMed1999

Tyrosine aminotransferase catalyzes the final step of methionine recycling in Klebsiella pneumoniae.

An aminotransferase which catalyzes the final step in methionine recycling from methylthioadenosine, the conversion of alpha-ketomethiobutyrate to methionine, has been purified from Klebsiella pneumoniae and characterized. The enzyme was found to be a homodimer of 45-kDa subunits, and it catalyzed methionine formation primarily using aromatic amino acids and glutamate as the amino donors. Histidine, leucine, asparagine, and arginine were also functional amino donors but to a lesser extent. The N-terminal amino acid sequence of the enzyme was determined and found to be almost identical to the N-terminal sequence of both the Escherichia coli and Salmonella typhimurium tyrosine aminotransferases (tyrB gene products). The structural gene for the tyrosine aminotransferase was cloned from K. pneumoniae and expressed in E. coli. The deduced amino acid sequence displayed 83, 80, 38, and 34% identity to the tyrosine aminotransferases from E. coli, S. typhimurium, Paracoccus denitrificans, and Rhizobium meliloti, respectively, but it showed less than 13% identity to any characterized eukaryotic tyrosine aminotransferase. Structural motifs around key invariant residues placed the K. pneumoniae enzyme within the Ia subfamily of aminotransferases. Kinetic analysis of the aminotransferase showed that reactions of an aromatic amino acid with alpha-ketomethiobutyrate and of glutamate with alpha-ketomethiobutyrate proceed as favorably as the well-known reactions of tyrosine with alpha-ketoglutarate and tyrosine with oxaloacetate normally associated with tyrosine aminotransferases. The aminotransferase was inhibited by the aminooxy compounds canaline and carboxymethoxylamine but not by substrate analogues, such as nitrotyrosine or nitrophenylalanine.

Amino Acid Sequence↗

PubMed1992

Ornithine delta-aminotransferase mutations in gyrate atrophy. Allelic heterogeneity and functional consequences.

Ornithine delta-aminotransferase is a nuclear-encoded mitochondrial matrix enzyme which catalyzes the reversible interconversion of ornithine and alpha-ketoglutarate to glutamate semialdehyde and glutamate. Inherited deficiency of ornithine delta-aminotransferase results in ornithine accumulation and a characteristic chorioretinal degeneration, gyrate atrophy of the choroid and retina. We have surveyed the ornithine delta-aminotransferase genes of gyrate atrophy patients for mutations. Using a variety of techniques, we discovered and molecularly characterized 21 newly recognized ornithine delta-aminotransferase alleles. We determined the consequences of these and three previously described mutations on ornithine delta-aminotransferase mRNA, antigen, and enzyme activity in cultured fibroblasts. The majority (20/24) of these alleles produce normal amounts of normally sized ornithine delta-aminotransferase mRNA. By contrast, only 2/24 had normal amounts of ornithine delta-aminotransferase antigen. Reproducing these mutations by site-directed mutagenesis and expressing the mutant ornithine delta-aminotransferase in Chinese hamster ovary cells confirms that several of these mutations inactivate ornithine delta-aminotransferase and cause gyrate atrophy in these patients.

Alleles↗

PubMed1987

Tyrosine aminotransferase gene expression in a temperature-sensitive adult rat liver cell line.

Regulation of tyrosine aminotransferase gene expression was studied in an adult rat hepatocyte line (RALA255-10G) which is temperature sensitive for the maintenance of the differentiated liver phenotype. Glucocorticoid hormones such as cortisol were necessary for expression of the aminotransferase gene. In the absence of these steroids, enzyme synthesis, activity, and mRNA accumulation were virtually abolished. In the presence of cortisol at 33 degrees C, RALA255-10G cells showed characteristics of malignant transformation and contained little tyrosine aminotransferase activity, synthesized low levels of this enzyme, and produced low levels of enzyme mRNA. At 40 degrees C, cells maintained in the presence of cortisol regained the normal, differentiated phenotype, and tyrosine aminotransferase synthesis and mRNA accumulation were greatly increased. This increase in aminotransferase synthesis paralleled the increase in the enzyme mRNA. However, after a temperature shift-up, tyrosine aminotransferase activity was increased only for the first 2 days, probably due to thermal inactivation of this enzyme at 40 degrees C. Dibutyryl cAMP alone was not sufficient to induce expression of the tyrosine aminotransferase gene, but it enhanced the induction caused by cortisol. Immunocytochemical studies revealed that the enhanced expression of the tyrosine aminotransferase gene at 40 degrees C and in the presence of cortisol or cortisol plus dibutyryl cAMP resulted from an increase in both the number of cells producing this enzyme and the quantity of tyrosine aminotransferase synthesized per cell.

Animals↗

PubMed1983

Pretranslational control of tyrosine aminotransferase synthesis by 8-bromo-cyclic AMP in H-4 rat hepatoma cells.

The H-4 rat hepatoma cell line grown in tissue culture was used as a model system to investigate the action of cAMP in tyrosine aminotransferase induction. An immunoprecipitation technique was used to quantitate the amount and the rate of synthesis of tyrosine aminotransferase; the level of mRNA coding for tyrosine aminotransferase was determined by in vitro translation of poly(A)+ RNA isolated from hepatoma cells. Our results demonstrated that 8-bromo-cAMP gave time-dependent and proportionate increases in the tyrosine aminotransferase activity, the amount of immunoprecipitable tyrosine aminotransferase, the rate of synthesis of tyrosine aminotransferase, and the level of mRNATAT in H-4 hepatoma cells. The time course of increase in mRNATAT preceded the increase in synthesis of tyrosine aminotransferase and was dependent on the continuous production of poly(A)+ RNA. Pretreatment of the cells with cordycepin completely abolished the 8-bromo-cAMP-evoked increase in mRNATAT activity. These results provided evidence that the primary action of cAMP in tyrosine aminotransferase induction is the increase of functional mRNATAT and that this increase can completely account for the increase in tyrosine aminotransferase activity.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1985

Coupled reaction of immobilized aspartate aminotransferase and malate dehydrogenase. A plausible model for the cellular behaviour of these enzymes.

To study the effect of facilitated diffusion of the intermediate metabolite, oxaloacetate, on the coupled reaction of aspartate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1) and malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37), these enzymes were co-immobilized on the surface of a collagen film. The kinetic properties of the immobilized enzymes were compared with those observed with the enzymes in solution. Since the reactions correspond to the cytosolic enzymes, they have been studied in the direction aspartate aminotransferase toward malate dehydrogenase. Coupled enzymes in solution showed classical behaviour. A lag-time was observed before they reached a steady state and this lag-time was dependent on the kinetic properties of the second enzyme, malate dehydrogenase. The same lag-time was observed when malate dehydrogenase in solution was coupled with aspartate aminotransferase bound to the film. When aspartate aminotransferase in solution was coupled with malate dehydrogenase bound to the collagen film, a very long lag-time was observed. Theoretical considerations showed that in the latter case, the lag-time was dependent on the kinetic properties of the second enzyme and the transport coefficient of the intermediate substrate through the boundary layer near the surface of the film. Then both enzymes were co-immobilized on the collagen film. The coupled activity of aspartate aminotransferase and malate dehydrogenase was compared for films with an activity ratio of 5 and 0.8. In both cases, a highly efficient coupling was observed. In the former case, where malate dehydrogenase was rate-limiting, 81% of this limiting activity was observed. In the latter case, aspartate aminotransferase was rate-limiting and 82% of its rate was obtained for the final product formation. The linear increase of product formation with time corresponded fairly well to the theoretical equations developed in the paper. To interpret these rate equations, one should assume that the intermediate substrate oxaloacetate formed by aspartate aminotransferase was used by malate dehydrogenase in the diffusion layer near the film, before diffusing in the bulk solution.

Animals↗

PubMed2000

Subjects positive for hepatitis C virus RNA with normal aminotransferase levels, a "trompe l'oeil" clinical picture?

BACKGROUND: Quite often subjects affected by chronic hepatitis C virus infection have no clinical signs of liver disease and serum aminotransferase values never go beyond the upper limit of normal. Yet these subjects, defined "asymptomatic HCV carriers", often have active viral replication and various degrees of histological damage. AIMS: To verify, in a population of antibody to hepatitis C virus carriers, if normal serum aminotransferase values in hepatitis C virus-RNA positive differed considerably from those in hepatitis C virus-RNA negative subjects. SUBJECTS/METHODS: We followed 24 anti-hepatitis C virus-positive subjects (15 hepatitis C virus-RNA positive and 9 negative) by measuring alanine and aspartate aminotransferase levels at 3-month intervals for a median of 40 months (range 6-77). RESULTS: Determinations resulted repeatedly and rigorously within the normal range in all participants. Alanine aminotransferase values were higher in hepatitis C virus-RNA positives than in negatives (mean +/- SD: 0.609+/-0.172 vs 0.434+/-0.153 times the upper limit of normal; p<O.001). Aspartate aminotransferase values in both groups reflected the same consistent difference (mean +/- SD: 0.652+/-0.170 vs 0.528+/-0.151 times the upper limit of normal, p<0.001). CONCLUSIONS: Our findings demonstrate that biochemical activity exists in asymptomatic hepatitis C virus-RNA-positive carriers, but it is so slight that serum aminotransferase levels do not go beyond the upper limit of normal. With the prevailing range of normality, serum aminotransferase determination is a helpful but not reliable marker of disease activity in chronic hepatitis C virus infection. The normal range for aminotransferases needs to be redefined in order to set up a more accurate diagnostic profile of subjects with chronic hepatitis C virus infection.

Adult↗

PubMed1981

Immunocytochemical localization of aspartate aminotransferase immunoreactivity in cochlear nucleus of the guinea pig.

There is substantial evidence supporting the role of aspartate or glutamate as the neurotransmitter of the auditory nerve. The concentration of aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1), an enzyme associated with the metabolism of these amino acids, is high in axons and terminals of the auditory nerve. Antibodies were raised against aspartate aminotransferase and used in immunocytochemical studies to determine its localization in the cochlear nucleus of the guinea pig. Indirect immunofluorescence techniques were used for light microscopic localization of aspartate aminotransferase-like immunoreactivity in normal guinea pigs and guinea pigs with auditory nerve lesions. Fluorescent rings of aspartate aminotransferase-like immunoreactivity were seen around spherical cells in the anteroventral cochlear nucleus. In animals with auditory nerve lesions, rings were no longer seen in the ipsilateral cochlear nucleus. Immunoreactivity was also seen on cells in the posteroventral cochlear nucleus and in auditory nerve fibers. Ultrastructural studies were done in the rostral anteroventral cochlear nucleus, using the peroxidase-antiperoxidase technique. Aspartate aminotransferase-like immunoreactivity was seen at axosomatic synapses on large spherical cells in terminals with the morphological characteristics of auditory nerve terminals. Other classes of terminals on the soma of large spherical cells showed no immunoreactivity. It was concluded that aspartate aminotransferase-like immunoreactivity is present in axons and terminals of the auditory nerve. These findings indicate that aspartate aminotransferase-like immunoreactivity may serve as a marker at terminals where aspartate or glutamate is a neurotransmitter.

Animals↗

PubMed1996

Irreversible inactivation of aspartate aminotransferase by 2-oxoglutaconic acid and its dimethyl ester.

Incubation of pig heart cytosolic aspartate aminotransferase (pyridoxal 5'-phosphate form) with 10 mM 2-oxoglutaconic acid dimethyl ester for 2 h at 25 degrees C (pH 7.0) results in slight inactivation (approximately 15%). However, incubation of the enzyme with glutamate, or prior conversion of the enzyme to the pyridoxamine 5'-phosphate form, results in more extensive inactivation. The inactivation of the enzyme by 2-oxoglutaconic acid dimethyl ester is most pronounced in the presence of both glutamate and alpha-ketoglutarate. N-Ethylmaleimide was previously shown to alkylate two surface cysteine residues (I and II) and to react syncatalytically with a third cysteine residue (III) of cytosolic pig heart aspartate aminotransferase [Birchmeier et al. (1973) J. Biol. Chem. 248, 1751-1759]. Alkylation of cysteine III results in inactivation of the enzyme, despite the fact that this residue is not essential for catalysis. The present results suggest that 2-oxoglutaconic acid dimethyl ester reacts with the enzyme in a similar fashion to that exhibited by N-ethylmaleimide. Some inactivation by alkylation of a susceptible group at the active site cannot be ruled out. However, the rate of inactivation of cytosolic pig heart aspartate aminotransferase is proportional to the concentration of 2-oxoglutaconic acid dimethyl ester up to a concentration of at least 40 mM, suggesting that the compound binds very poorly to the active site or that alkylation at the active site is slow compared with syncatalytic alkylation of cysteine III. The t 1/2 for inactivation of pig heart cytosolic aspartate aminotransferase by 40 mM 2-oxoglutaconic acid dimethyl ester (in the presence of 10 mM L-glutamate, pH 7.2, 25 degrees C) is 9 min. Incubation of cytosolic pig heart aspartate aminotransferase with 10 mM 2-oxoglutaconate for 2 h (25 degrees C, pH 7.2) results in significant inactivation (approximately 30%). The enzyme is protected against inactivation by the presence of alpha-ketoglutarate, but glutamate enhances the inactivation. These findings suggest that 2-oxoglutaconate is an active site-directed inhibitor. The binding of 2-oxoglutaconate to the enzyme exhibits saturation kinetics (K1 approximately 2 mM), but the rate of inactivation is slow (limiting rate constant for inactivation in the presence of L-glutamate approximately 0.01 min-1; pH 6.0, 25 degrees C; t 1/2 max approximately 70 min). This finding suggests that 2-oxoglutaconate does not readily react in a syncatalytic fashion with cysteine III. Possibly, the two negative charges of 2-oxoglutaconate do not allow ready approach to cysteine III. Rather, the findings suggest that 2-oxoglutaconate binds at the active site of the pyridoxal 5'-phosphate form of the enzyme as an affinity labeling reagent. However, the increased rate of 2-oxoglutaconate-induced inactivation in the presence of glutamate suggests that this unsaturated alpha-keto acid also exhibits the properties of a kcat inhibitor. 2-Oxoglutaconate inactivates aspartate aminotransferase in cytosolic and mitochondrial fractions of rat kidney and purified pig heart alanine aminotransferase. Injection of 2-oxoglutaconate into mice results in inhibition of kidney aspartate aminotransferase. 2-Oxoglutaconate is a substrate of glutamate dehydrogenase. The kinetic constants are similar to those obtained with alpha-ketoglutarate. The results suggest that unsaturated alpha-keto acids and their esters may be useful probes for the study of alpha-keto acid-utilizing enzymes.

Animals↗

PubMed2006

Values and limitations of serum aminotransferases in clinical trials of nonalcoholic steatohepatitis.

BACKGROUND/AIMS: Choosing endpoints in nonalcoholic steatohepatitis (NASH) trials is challenging because of the lack of validated surrogates and the trade-off between accuracy and invasiveness. In this study, we assessed diagnostic accuracy of serum aminotransferase changes in predicting histological changes in NASH trials. METHODS: We conducted a longitudinal cohort study by using 102 participants in ursodeoxycholic acid-NASH trial who had both baseline and 2-year liver biopsy and multiple measurements of serum aminotransferases. We calculated rates of alanine aminotransferase (ALT) [or aspartate aminotransferase (AST)] change as slopes of linear regression over 2 years (IU/l/month) and changes in each histological feature as differences in Brunt's scores of two biopsies in each individual. RESULTS: Rate of aminotransferase change correlated with changes in inflammation and fibrosis, but not steatosis and only with change in inflammation in multivariable analysis. In each histological feature, changes were inversely correlated with baseline histological grade. In predicting improvement of inflammation, areas under the receiver-operating characteristic curve of aminotransferase information alone were 0.72 for ALT and 0.73 for AST and were improved to 0.88 and 0.89, respectively, when baseline histology were taking account of. CONCLUSIONS: Serum aminotransferase changes could be useful as surrogates in screening therapies for NASH in clinical trials with appropriate consideration of baseline aminotransferase and histology.

Adult↗

PubMed1977

Escherichia coli mutants deficient in the aspartate and aromatic amino acid aminotransferases.

Two new mutations are described which, together, eliminate essentially all the aminotransferase activity required for de novo biosynthesis of tyrosine, phenylalanine, and aspartic acid in a K-12 strain of Escherichia coli. One mutation, designated tyrB, lies at about 80 min on the E. coli map and inactivates the "tyrosine-repressible" tyrosine/phenylalanine aminotransferase. The second mutation, aspC, maps at about 20 min and inactivates a nonrespressible aspartate aminotransferase that also has activity on the aromatic amino acids. In ilvE- strains, which lack the branched-chain amino acid aminotransferase, the presence of either the tyrosine-repressible aminotransferase or the aspartate aminotransferase is sufficient for growth in the absence of exogenous tyrosine, phenylalanine, or aspartate; the tyrosine-repressible enzyme is also active in leucine biosynthesis. The ilvE gene product alone can reverse a phenylalanine requirement. Biochemical studies on extracts of strains carrying combinations of these aminotransferase mutations confirm the existence of two distinct enzymes with overlapping specificities for the alpha-keto acid analogues of tyrosine, phenylalanine, and aspartate. These enzymes can be distinguished by electrophoretic mobilities, by kinetic parameters using various substrates, and by a difference in tyrosine repressibility. In extracts of an ilvE- tyrB- aspC- triple mutant, no aminotransferase activity for the alpha-keto acids of tyrosine, phenylalanine, or aspartate could be detected.

Aspartate Aminotransferases↗

PubMed1994

Low serum aminotransferase activity in patients undergoing regular hemodialysis.

Of 150 patients undergoing regular hemodialysis (HD), 14 (9.3%) and 12 (8.0%), respectively, showed low serum activity of aspartate aminotransferase (AST) and alanine aminotransferase (ALT). An investigation was conducted to elucidate the underlying mechanisms in 20 patients with low serum AST and/or ALT activity. Fifty-five percent of the patients with low aminotransferase activity manifested serum levels of pyridoxal phosphate (PLP) that were lower than normal. Serum PLP levels correlated neither with AST nor with ALT activity. Oral administration of vitamin B6 to the cases with low aminotransferase activity resulted in an increase in pre-hemodialysis aminotransferase activity. Addition of vitamin B6 in vitro to the sera from the patients with low aminotransferase activity did not increase the values when the added vitamin B6 was within the physiological range, but did increase when added in larger (pharmacological) amounts. However, aminotransferase activity increased, but PLP levels remained unchanged when these values were compared before and after HD. On the other hand, guanase being within the normal range in all cases studied, did not change after HD. Although our study does not correlate with vitamin B6 deficiency, but rather with some uremic substance(s) which interfere(s) with the enzyme reaction as a cause of low aminotransferase activity, the fact that less than 10% of our patients showed low AST and/or ALT points to the latter possibility, suggesting the need of further study.

Adult↗

PubMed2006

Review article: management of patients with chronic hepatitis C virus infection and "normal" alanine aminotransferase activity.

BACKGROUND: Hepatitis C virus infection, a major cause of chronic liver disease, occurs with normal serum alanine aminotransferase activity in approximately 25% of patients. These patients have historically remained untreated but substantial evidence indicates liver damage, progression of disease and impaired quality of life in some individuals. AIM: To review the current management of patients with chronic hepatitis C and normal alanine aminotransferase activity. METHODS: This review represents the summary of discussions at a Clinical Workshop with a comprehensive literature searching of available databases (PubMed and Embase). RESULTS: Current limits defining normal serum alanine aminotransferase activity are not representative of a "healthy" status. Most patients with hepatitis C and normal alanine aminotransferase levels have histologically proven liver damage that, although generally mild, may be significant (> or =F2) in up to 20% of patients and progresses at approximately 50% of the rate in patients with elevated alanine aminotransferase levels. Some patients have persistently normal alanine aminotransferase activity and may have a more benign outcome, but a significant proportion (> or =20%) experience periods of increased serum alanine aminotransferase activity which may be associated with enhanced disease progression. CONCLUSIONS: A treatment approach that considers host and virus-related variables and optimizes patient and cost benefits may therefore provide more effective management of patients with chronic hepatitis C and normal alanine aminotransferase activity.

Alanine Transaminase↗

PubMed1990

Cloning and sequence of the Salmonella typhimurium hemL gene and identification of the missing enzyme in hemL mutants as glutamate-1-semialdehyde aminotransferase.

Salmonella typhimurium forms the heme precursor delta-aminolevulinic acid (ALA) exclusively from glutamate via the five-carbon pathway, which also occurs in plants and some bacteria including Escherichia coli, rather than by ALA synthase-catalyzed condensation of glycine and succinyl-coenzyme A, which occurs in yeasts, fungi, animal cells, and some bacteria including Bradyrhizobium japonicum and Rhodobacter capsulatus. ALA-auxotrophic hemL mutant S. typhimurium cells are deficient in glutamate-1-semialdehyde (GSA) aminotransferase, the enzyme that catalyzes the last step of ALA synthesis via the five-carbon pathway. hemL cells transformed with a plasmid containing the S. typhimurium hemL gene did not require ALA for growth and had GSA aminotransferase activity. Growth in the presence of ALA did not appreciably affect the level of extractable GSA aminotransferase activity in wild-type cells or in hemL cells transformed with the hemL plasmid. These results indicate that GSA aminotransferase activity is required for in vivo ALA biosynthesis from glutamate. In contrast, extracts of both wild-type and hemL cells had gamma,delta-dioxovalerate aminotransferase activity, which indicates that this reaction is not catalyzed by GSA aminotransferase and that the enzyme is not encoded by the hemL gene. The S. typhimurium hemL gene was sequenced and determined to contain an open reading frame of 426 codons encoding a 45.3-kDa polypeptide. The sequence of the hemL gene bears no recognizable similarity to the hemA gene of S. typhimurium or E. coli, which encodes glutamyl-tRNA reductase, or to the hemA genes of B. japonicum or R. capsulatus, which encode ALA synthase. The predicted hemL gene product does show greater than 50% identity to barley GSA aminotransferase over its entire length. Sequence similarity to other aminotransferases was also detected.

Amino Acid Sequence↗

PubMed1976

Purification and properties of two aromatic aminotransferases in Bacillus subtilis.

Two enzymes which transaminate tyrosine and phenylalanine in Bacillus subtilis were each purified over 200-fold and partially characterized. One of the enzymes, termed histidinol phosphate aminotransferase, is also active with imidazole acetyl phosphate as the amino group recipient. Previous studies have shown that mutants lacking this enzyme require histidine for growth. Mutants in the other enzyme termed aromatic aminotransferase are prototrophs. Neither enzyme is active on any other substrate involved in amino acid synthesis. The two enzymes can be distinguished by a number of criteria. Gel filtration analysis indicate the aromatic and histidinol phosphate aminotransferases have molecular weights of 63,500 and 33,000, respectively. Histidinol phosphate aminotransferase is heat-sensitive, whereas aromatic aminotransferase is relatively heat-stable, particularly in the presence of alpha-ketoglutarate. Both enzymes display typical Michaelis-Menten kinetics in their rates of reaction. The two enzymes have similar pH optima and employ a ping-pong mechanism of action. The Km values for various substrates suggest that histidinol phosphate aminotransferase is the predominant enzyme responsible for the transamaination reactions in the synthesis of tyrosine and phenylalanine. This enzyme has a 4-fold higher affinity for tyrosine and phenylalanine than does the aromatic aminotransferase. Competitive substrate inhibition was observed between tyrosine, phenylalanine, and histidinol phosphate for histidinol phosphate aminotransferase. The significance of the fact that an enzyme of histidine synthesis plays an important role in aromatic amino acid synthesis is discussed.

Bacillus subtilis↗

PubMed1992

Identification of mitochondrial branched chain aminotransferase and its isoforms in rat tissues.

The tissue distribution and subcellular location of branched chain aminotransferase was analyzed using polyclonal antibodies against the enzyme purified from rat heart mitochondria (BCATm). Immunoreactive proteins were visualized by immunoblotting. The antiserum recognized a 41-kDa protein in the 100,000 x g supernatant from a rat heart mitochondrial sonicate. The 41-kDa protein was always present in mitochondria which contained branched chain aminotransferase activity, skeletal muscle, kidney, stomach, and brain, but not in cytosolic fractions. In liver mitochondria, which have very low levels of branched chain aminotransferase activity, the 41-kDa protein was not present. However, two immunoreactive proteins of slightly higher molecular masses were identified. These proteins were located in hepatocytes. The 41-kDa protein was present in fetal liver mitochondria but not in liver mitochondria from 5-day neonates. Thus disappearance of the 41-kDa protein coincided with the developmental decline in liver branched chain aminotransferase activity. Two-dimensional immunoblots of isolated BCATm immunocomplexes showed that the liver immunoreactive proteins were clearly different from the heart and kidney proteins which exhibited identical immunoblots. Investigation of BCATm in subcellular fractions prepared from different skeletal muscle fiber types revealed that branched chain aminotransferase is exclusively a mitochondrial enzyme in skeletal muscles. Although total detergent-extractable branched chain aminotransferase activity was largely independent of fiber type, branched chain aminotransferase activity and BCATm protein concentration were highest in mitochondria prepared from white gastrocnemius followed by mixed skeletal muscles with lowest activity and protein concentration found in soleus mitochondria. These quantitative differences in mitochondrial branched chain aminotransferase activity and enzyme protein content suggest there may be differential expression of BCATm in different muscle fiber types.

Aging↗

PubMed1978

Increase in hepatic tyrosine aminotransferase mRNA during enzyme induction by N6,O2'-dibutyryl cyclic AMP.

Tyrosine aminotransferase mRNA was quantitated by translation in a cell-free system derived from wheat germ followed by specific immunoprecipitation of the newly synthesized enzyme subunit. Hepatic poly(A)-containg RNA prepared from rats treated for 4 h with N6, O2'-dibutyryl cyclic AMP and theophylline was approximately 5.6 times more active in directing the synthesis of the tyrosine aminotransferase subunit relative to untreated controls. The overall template activity of the RNA prepared from control and cyclic AMP-treated animals was virtually identical, demonstrating that the cyclic nucleotide effect was specific for the tyrosine aminotransferase mRNA. At all times, after a single injection of dibutyryl cyclic AMP and theophylline, the increase in hepatic enzyme activity was accompanied by corresponding induction in the level of functional tyrosine aminotransferase mRNA. Other inducers of tyrosine aminotransferase, such as glucagon and hydrocortisone, also increased the level of tyrosine aminotransferase mRNA in proportion to their effect on enzyme activity. The RNA polymerase II inhibitor, alpha-amanitin, completely blocked the dibutyryl cyclic AMP-mediated increase in tyrosine aminotransferase mRNA activity. These studies demonstrate that, in intact animals, the induction of tyrosine aminotransferase activity by dibutyryl cyclic AMP can be completely accounted for by a corresponding increase in the level of functional mRNA coding for the enzyme.

Animals↗