Purification and properties of aromatic amino acid aminotransferase from Klebsiella aerogenes.

We describe the complete purification of aromatic aminotransferase I, the enzyme responsible for the ability of Klebsiella aerogenes to use tryptophan and phenylalanine as sole sources of nitrogen, as well as the partial purification of aromatic aminotransferase IV. An examination of the properties of these enzymes revealed that aminotransferase I had much greater affinity for the aromatic amino acids than aminotransferase IV, explaining the essential role of aminotransferase I in the utilization of exogenously supplied aromatic amino acids. The properties of aminotransferase IV suggest that this enzyme is actually an aspartate aminotransferase (EC 2.6.1.1), corresponding to the product of the aspC gene of Escherichia coli.     

Aspartate: 2-oxoglutarate aminotransferase from trichomonas vaginalis. Identity of aspartate aminotransferase and aromatic amino acid aminotransferase.

Aspartate: 2-oxoglutarate aminotransferase from the anaerobic protozoon Trichomonas vaginalis was purified to homogeneity and characterized. It is a dimeric protein of overall Mr approx. 100000. Only a single isoenzyme was found in T. vaginalis. The overall molecular and catalytic properties have features in common with both the vertebrate cytoplasmic and mitochondrial isoenzymes. The purified aspartate aminotransferase from T. vaginalis showed very high rates of activity with aromatic amino acids as donors and 2-oxoglutarate as acceptor. This broad-spectrum activity was restricted to aromatic amino acids and aromatic 2-oxo acids, and no significant activity was seen with other common amino acids, other than with the substrates and products of the aspartate: 2-oxoglutarate aminotransferase reaction. Co-purification and co-inhibition, by the irreversible inhibitor gostatin, of the aromatic amino acid aminotransferase and aspartate aminotransferase activities, in conjunction with competitive substrate experiments, strongly suggest that a single enzyme is responsible for both activities. Such high rates of aromatic amino acid aminotransferase activity have not been reported before in eukaryotic aspartate aminotransferase.     

Characterization of an aromatic amino acid aminotransferase from Rhizobium leguminosarum biovar trifolii.

The most abundant aromatic amino acid aminotransferase of Rhizobium leguminosarum biovar trifolii was partially purified. The molecular mass of the enzyme was estimated to be 53 kDa by gel filtration. The enzyme transaminated aromatic amino acids and histidine. It used aromatic keto acids and alpha-ketoglutaric and oxalacetic acids as amino-group acceptors. The optimum temperature was 35 degrees C. Using phenylalanine and alpha-ketoglutaric acid as substrates the activation energy was 46.2 kJ.mol-1 and for the couple tryptophan:alpha-ketoglutaric acid it was 70.3 kJ.mol-1. The optimum pH was different for each substrate: 7.3 for phenylalanine, 7.9 for histidine and 8.7 for tryptophan.     

Cloning and sequencing of the gene coding for aspartate aminotransferase from the thermoacidophilic archaebacterium Sulfolobus solfataricus

The gene coding for aspartate aminotransferase (EC 2.6.1.1) has been cloned from the extreme thermoacidophilic archaebacterium Sulfolobus solfataricus strain MT4. Partial sequence data obtained directly from the purified protein and from the two cyanogen-bromide-generated peptides confirm the primary structure of aspartate aminotransferase inferred from the nucleotide sequence of its gene. A comparison of the enzyme with other aminotransferases revealed an interesting similarity with tyrosine aminotransferase from rat liver (EC 2.6.1.5) and allowed some tentative assignments of the residues implied in the catalysis. The aspartate aminotransferase gene-flanking regions were compared to those of other archaebacterial genes already described in the literature with the aim of identifying potential regulatory sites.     

Role of the Escherichia coli aromatic amino acid aminotransferase in leucine biosynthesis.

Strains of Escherichia coli that lack the branched-chain amino acid amino-transferase because of mutations in the ilvE gene had no growth requirement for leucine when the cells contained the aromatic amino acid aminotransferase that is the product of the tyrB gene. The presence of leucine increased the generation time of these cells and decreased the specific activity of the aromatic amino acid aminotransferase. It is concluded that this enzyme functions efficiently in leucine biosynthesis and can be repressed by leucine as well as by tyrosine.     

Purification and properties of the aromatic amino acid aminotransferase from gramicidin S-producing Bacillus brevis

The aromatic amino acid aminotransferase was purified to a homogenous state from a gramicidin S-producing strain of Bacillus brevis. The enzyme shows a molecular weight of about 71,000 on gel-filtration. The subunit molecular weight is about 35,000 as determined by sodium dodecyl sulfate gel electrophoresis, indicating that the enzyme is a dimer. The enzyme exhibits absorption maxima near 425 and 330 nm at neutral pH. One mole of pyridoxal phosphate is bound per subunit. The enzyme has amino donor specificity for aromatic amino acids, L-phenylalanine, L-tyrosine, and L-tryptophan, and utilizes 2-oxoglutarate as the amino acceptor. This enzyme activity was separated from both the aspartate aminotransferase activity and the branched chain amino acid aminotransferase activity by chromatography on DEAE-Sephadex.     

Mapping of the aspartate and aromatic amino acid aminotransferase genes tyrB and aspC.

Co-transduction experiments using P1-mediated reciprocal and three-factor crosses have been used to map two mutations affecting the aspartate and aromatic amino acid aminotransferases of Escherichia coli. tyrB-, which inactivates the tyrosine-repressible component of these activities is co-transducible with metA and malB; the gene order is metA-malB-tyrB. aspC-, which inactivates the nonrepressible aminotransferase with high activity for aspartate, maps between and is co-transducible with serC and pyrD.     

Utilization of aromatic amino acids as nitrogen sources in Brevibacterium linens: an inducible aromatic amino acid aminotransferase

The first step of the utilization of the aromatic amino acids as sole nitrogen sources by Brevibacterium linens strain 47 was found to be a transamination. The deaminated metabolites of the amino acids were detected in culture supernatants, and the enzyme activity was identified in cell free extracts. The cells contained increased aromatic amino acid aminotransferase activities on growth on the aromatic amino acids as sole nitrogen sources. Two aromatic aminotransferases (AT-I and AT-II) were separated upon diethylaminoethyl-Trisacryl M column chromatography of cell free extracts. Only AT-I was responsible for the increased level of aromatic amino acid aminotransferase activity of induced cells. The results suggested a catabolic role of AT-I in vivo.Abbreviations DNP dinitrophenyl - HPLC high performance liquid chromatography - PLP pyridoxal-5-phosphate     

A branched-chain amino acid aminotransferase from the rumen ciliate genus Entodinium.

A branched-chain amino acid aminotransferase was extracted from rumen ciliates of the genus Entodinium and was partially purified by Sephadex G-200, DEAE-cellulose and DEAE-Sephasex A-50 column chromatography. The purified enzyme was active only with leucine, isoleucine and valine, and required pyridoxal phosphate as cofactor. The amino acids competed with each other as substrates. The enzyme had optimal activity at pH 6.0 in phosphate buffer. The Km values for the substrates and cofactor are as follows: 1.66 for leucine; 0.90 for isoleucine; 0.79 for valine; 0.29 mM for alpha-ketoglutarate; and 0.1 muM for pyridoxal phosphate. Enzyme activity was inhibited by rho-chloromercuribenzoate and HgCl2. Gel filtration indicated the enzyme to have a molecular weight of 34,000.     

The substrate activation process in the catalytic reaction of Escherichia coli aromatic amino acid aminotransferase

Aromatic amino acid aminotransferase is active toward both aromatic and dicarboxylic amino acids, and the mechanism for this dual substrate recognition has been an issue in the enzymology of this enzyme. Here we show that, in the reactions with aromatic and dicarboxylic ligands, the pK(a) of the Schiff base formed between the coenzyme pyridoxal 5'-phosphate and Lys258 or the substrate increases successively from 6.6 in the unliganded enzyme to approximately 8.8 in the Michaelis complex and to >10.5 in the external Schiff base complex. Mutations of Arg292 and Arg386 to Leu, which mimic neutralization of the positive charges of the two arginine residues by the ligand carboxylate groups, increased the Schiff base pK(a) by 0.1 and 0.7 unit, respectively. In contrast to these moderate effects of the Arg mutations, the cleavage of the Lys258 side chain of the Schiff base, which was brought about by preparing a mutant enzyme in which Lys258 was changed to Ala and the Schiff base was reconstituted with methylamine, produced the Schiff base pK(a) value of 10.2, that being 3.6 units higher than that of the wild-type enzyme. The observation indicates that the Schiff base pK(a) in the enzyme is lowered by the torsion around the C4-C4' axis of the Schiff base and suggests that the pK(a) is mainly controlled by changing the torsion angle during the course of catalysis. This mechanism, first observed for the reaction of aspartate aminotransferase with aspartate [Hayashi, H., Mizuguchi, H., and Kagamiyama, H. (1998) Biochemistry 37, 15076-15085], does not require the electrostatic contribution from the omega-carboxylate group of the substrate, and can explain why in aromatic amino acid aminotransferase the aromatic substrates can increase the Schiff base pK(a) during catalysis to the same extent as the dicarboxylic substrates. This is the first example in which the torsion pK(a) coupling of the pyridoxal 5'-phosphate Schiff base has been demonstrated in pyridoxal enzymes other than aspartate aminotransferase, and suggests the generality of the mechanism in the catalysis of aminotransferases related to aspartate aminotransferase.     

Partial purification and characterisation of an aromatic amino acid aminotransferase from mung bean (Vigna radiata L. Wilczek)

An aromatic amino acid aminotransferase (aromAT) was purified over 33 000-fold from the shoots and primary leaves of mung beans (Vigna radiata L. Wilczek). The enzyme was purified by ammonium sulfate precipitation, gel filtration and anion exchange followed by fast protein liquid chromatography using Mono Q and Phenylsuperose. The relative amino transferase activities using the most active amino acid substrates were: tryptophan 100, tyrosine 83 and phenylalanine 75, withK _m values of 0.095, 0.08 and 0.07 mM, respectively. The enzyme was able to use 2-oxoglutarate, oxaloacetate and pyruvate as oxo acid substrates at relative activities of 100, 128 and 116 andK _m values of 0.65, 0.25 and 0.24 mM, respectively. In addition to the aromatic amino acids the enzyme was able to transaminate alanine, arginine, aspartate, leucine and lysine to a lesser extent. The reverse reactions between glutamate and the oxo acids indolepyruvate and hydroxyphenylpyruvate occurred at 30 and 40% of the forward reactions of tryptophan and tyrosine, withK _m, values of 0.1 and 0.8 mM, respectively. The enzyme was not inhibited by indoleacetic acid, although -naphthaleneacetic acid did inhibit slightly. Addition of the cofactor pyridoxal phosphate only slightly increased the activity of the purified enzyme. The aromAT had a molecular weight of 55–59 kDa. The possible role of the aromAT in the biosynthesis of indoleacetic acid is discussed.Abbreviations AAT aspartate aminotransferase - aromAT aromatic amino acid aminotransferase - FPLC fast protein liquid chromatography - IPyA indolepyruvate - OHPhPy hydroxyphenylpyruvate - PLP pyridoxal phosphate - TAT tryptophan aminotransferase     

Expression of a synthetic gene coding for the amino acid sequence of Clostridium pasteurianum rubredoxin.

A synthetic gene based on the published amino acid sequence for Clostridium pasteurianum rubredoxin was constructed, cloned in Escherichia coli 71/18 and expressed using the T7 RNA polymerase/promoter system in E. coli HMS273. UV/visible spectroscopy and metal analyses indicated that the as-isolated synthetic gene product is a mixture of holo-(i.e. iron-containing) rubredoxin and zinc-substituted rubredoxin, with the latter amounting to approximately 70% of the total rubredoxin. The UV/visible absorption and resonance Raman spectra of the cloned holorubredoxin are characteristic of the native rubredoxin-type iron site. N-terminal amino acid sequencing suggests that the gene product consists of at least three polypeptide species with the initial sequences (approximate relative abundances): Met-Met-Lys-... (63%), blocked (30%) and Met-Lys-... (7%). The blocked portion presumably consists of a mixture of nMet-Met-Lys-... and nMet-Lys-..., where nMet represents an amino-blocked methionine residue.     

Cloning and characterization of a novel fold-type I branched-chain amino acid aminotransferase from the hyperthermophilic archaeon Thermococcus sp. CKU-1

We successfully cloned a novel branched-chain amino acid aminotransferase (Ts-BcAT; EC 2.6.1.42) gene from the Thermococcus sp. CKU-1 genome and expressed it in the soluble fraction of Escherichia coli Rosetta (DE3) cells. Ts-BcAT is a homodimer with an apparent molecular mass of approximately 92 kDa. The primary structure of Ts-BcAT showed high homology with the fold-type I, subgroup I aminotransferases, but showed little homology with BcATs known to date, i.e., those of Escherichia coli and Salmonella typhimurium, which belong to the fold-type IV, subgroup III aminotransferases. The maximum enzyme activity of Ts-BcAT was detected at 95 °C, and Ts-BcAT did not lose any enzyme activity, even after incubation at 90 °C for 5 h. Ts-BcAT was active in the pH range from 4.0 to 11.0, the optimum pH was 9.5, and the enzyme was stable between pH 6 and 7. The exceptionally low pK _a of the nitrogen atom in the Lys258 ε-amino group in the internal aldimine bond of Ts-BcAT was determined to be 5.52 ± 0.05. Ts-BcAT used 21 natural and unnatural amino acids as a substrate in the overall transamination reaction. l-Leucine and other aliphatic amino acids are efficient substrates, while polar amino acids except glutamate were weak substrates. Phylogenetic analysis revealed that Ts-BcAT is a novel fold-type I, subgroup I branched-chain aminotransferase.     

The amino acid sequence of cytosolic aspartate aminotransferase from human liver.

1. The cytosolic aspartate aminotransferase was purified from human liver. 2. The isoenzyme contains four cysteine residues, only one of which reacts with 5,5'-dithiobis-(2-nitrobenzoic acid) in the absence of denaturing agents. 3. The amino acid sequence of the isoenzyme is reported, as determined from peptides produced by digestion with trypsin and with CNBr, and from sub-digestion of some of these peptides with Staphylococcus aureus V8 proteinase. 4. The isoenzyme shares 48% identity of amino acid sequence with the mitochondrial form from human heart. 5. Comparisons of the amino acid sequences of all known mammalian cytosolic aspartate aminotransferases and of the same set of mitochondrial isoenzymes are reported. The results indicate that the cytosolic isoenzymes have evolved at about 1.3 times the rate of the mitochondrial forms. 6. The time elapsed since the cytosolic and mitochondrial isoenzymes diverged from a common ancestral protein is estimated to be 860 x 10(6) years. 7. Experimental details and confirmatory data for the results presented here are given in a supplementary paper that has been deposited as a Supplementary Publication SUP 50158 (25 pages) at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1990) 265, 5.     

Cloning of the transketolase gene and the effect of its dosage on aromatic amino acid production in Corynebacterium glutamicum

Transketolase is a key enzyme of the nonoxidative pentose phosphate pathway. The effect of its overexpression on aromatic amino acid production was investigated in Corynebacterium glutamicum, a typical amino-acid-producing organism. For this purpose, the transketolase gene of the organism was cloned on the basis of its ability to complement a C. glutamicum transketolase mutant with pleiotropically shikimic-acid-requiring, ribose- and gluconic-acid-negative phenotype. The gene was shown by deletion mapping and complementation analysis to be located in a 3.2-kb XhoI-SalI fragment of the genome. Amplification of␣the gene by use of low-, middle-, and high-copy-number vectors in a C. glutamicum strain resulted in overexpression of transketolase activities as well as a␣protein of approximately 83kDa in proportion to the copy numbers. Introduction of the plasmids into a tryptophan and lysine co-producer resulted in copy-dependent increases in tryptophan production along with concomitant decreases in lysine production. Furthermore, the presence of the gene in high copy numbers enabled tyrosine, phenylalanine and tryptophan producers to accumulate 5%–20% more aromatic amino acids. These results indicate that overexpressed transketolase activity operates to redirect the glycolytic intermediates toward the nonoxidative pentose phosphate pathway in vivo, thereby increasing the intracellular level of erythrose 4-phosphate, a precursor of aromatic biosynthesis, in the aromatic-amino-acid-producing C. glutamicum strains. Received: 27 July 1998 / Received last revision: 12 October 1998 / Accepted: 24 October 1998     

Cloning of the genes for excitatory amino acid receptors.

Glutamate is the major excitatory neurotransmitter in the mammalian brain, with receptors on every neuron in the central nervous system; it has major roles in fast synaptic transmission and in the establishment of certain forms of memory. More than 20 years ago Olney and his colleagues described the 'Excitotoxic Hypothesis' which postulates that, in addition to its normal function in the healthy brain, glutamate can kill neurons by prolonged, receptor-mediated depolarization resulting in irreversible disturbances in ion homeostasis. Therefore, glutamate is a two-edged sword; in certain undefined, adverse conditions it undergoes a transition from neurotransmitter to neurotoxin. Its toxicity has been implicated in the death of neurons in ischemia, epilepsy, and the neurodegenerative disorders such as Alzheimer's, Huntington's, and Parkinson's disease. Recent advances in the molecular cloning of the genes for the glutamate family of receptors has revealed a plethora of receptor subtypes and an unexpected level of complexity in the mechanisms of receptor expression and function.