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

1985 ◽  
Vol 232 (3) ◽  
pp. 689-695 ◽  
Author(s):  
P N Lowe ◽  
A F Rowe
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Aspartate Aminotransferase ◽  
Aromatic Amino Acid ◽  
Trichomonas Vaginalis ◽  
Aromatic Amino Acids ◽  
Significant Activity ◽  
Irreversible Inhibitor ◽  
Competitive Substrate ◽  
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.

scholarly journals Histidine Degradation via an Aminotransferase Increases the Nutritional Flexibility of Candida glabrata

2014 ◽  
Vol 13 (6) ◽  
pp. 758-765 ◽  
Author(s):  
Sascha Brunke ◽  
Katja Seider ◽  
Martin Ernst Richter ◽  
Sibylle Bremer-Streck ◽  
Shruthi Ramachandra ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Aromatic Amino Acid ◽  
Candida Glabrata ◽  
Genomic Structure ◽  
Aromatic Amino Acids ◽  
Sole Source ◽  
Upstream Region ◽  
Content Type ◽  
Aromatic Amino Acid Aminotransferase

ABSTRACTThe ability to acquire nutrients during infections is an important attribute in microbial pathogenesis. Amino acids are a valuable source of nitrogen if they can be degraded by the infecting organism. In this work, we analyzed histidine utilization in the fungal pathogen of humansCandida glabrata. Hemiascomycete fungi, likeC. glabrataorSaccharomyces cerevisiae, possess no gene coding for a histidine ammonia-lyase, which catalyzes the first step of a major histidine degradation pathway in most other organisms. We show thatC. glabratainstead initializes histidine degradation via the aromatic amino acid aminotransferase Aro8. AlthoughARO8is also present inS. cerevisiaeand is induced by extracellular histidine, the yeast cannot use histidine as its sole nitrogen source, possibly due to growth inhibition by a downstream degradation product. Furthermore,C. glabratarelies only on Aro8 for phenylalanine and tryptophan utilization, sinceARO8, but not its homologueARO9, was transcriptionally activated in the presence of these amino acids. Accordingly, anARO9deletion had no effect on growth with aromatic amino acids. In contrast, inS. cerevisiae,ARO9is strongly induced by tryptophan and is known to support growth on aromatic amino acids. Differences in the genomic structure of theARO9gene betweenC. glabrataandS. cerevisiaeindicate a possible disruption in the regulatory upstream region. Thus, we show that, in contrast toS. cerevisiae,C. glabratahas adapted to use histidine as a sole source of nitrogen and that the aromatic amino acid aminotransferase Aro8, but not Aro9, is the enzyme required for this process.

scholarly journals Molecular analysis of the role of two aromatic aminotransferases and a broad-specificity aspartate aminotransferase in the aromatic amino acid metabolism ofPyrococcus furiosus

Archaea ◽  
2002 ◽  
Vol 1 (2) ◽  
pp. 133-141 ◽  
Author(s):  
Donald E. Ward ◽  
William M. de Vos ◽  
John van der Oost
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Aspartate Aminotransferase ◽  
Pyrococcus Furiosus ◽  
Aromatic Amino Acids ◽  
Significant Activity ◽  
Transamination Reaction ◽  
Genes Encoding ◽  
Branched Chain ◽  
Aromatic Amino Acid Metabolism

The genes encoding aromatic aminotransferase II (AroAT II) and aspartate aminotransferase (AspAT) fromPyrococcus furiosushave been identified, expressed inEscherichia coliand the recombinant proteins characterized. The AroAT II enzyme was specific for the transamination reaction of the aromatic amino acids, and uses α-ketoglutarate as the amino acceptor. Like the previously characterized AroAT I, AroAT II has highest efficiency for phenylalanine (kcat/Km= 923 s–1mM–1). Northern blot analyses revealed that AroAT I was mainly expressed when tryptone was the primary carbon and energy source. Although the expression was significantly lower, a similar trend was observed for AroAT II. These observations suggest that both AroATs are involved in amino acid degradation. Although AspAT exhibited highest activity with aspartate and α-ketoglutarate (kcat~105 s–1), it also showed significant activity with alanine, glutamate and the aromatic amino acids. With aspartate as the amino donor, AspAT catalyzed the amination of α-ketoglutarate, pyruvate and phenylpyruvate. No activity was detected with either branched-chain amino acids or α-keto acids. The AspAT gene (aspC) was expressed as a polycistronic message as part of thearooperon, with expression observed only when the aromatic amino acids were absent from the growth medium, indicating a role in the biosynthesis of the aromatic amino acids.

Selective Chemical Deuteration of Aromatic Amino Acids: A Retrospective

1997 ◽  
Author(s):  
K.S. Matthews ◽  
R. Matthews
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Aromatic Amino Acid ◽  
Protein Hydrolysate ◽  
Aromatic Amino Acids ◽  
Cellular Protein ◽  
Target Protein ◽  
Chemical Methods ◽  
Lactose Repressor ◽  
Selective Deuteration

In 1970 when we began post-doctoral work in the laboratory of Professor Oleg Jardetzky, selective deuteration of proteins to limit the number of protons present in the system for subsequent analysis was a newly developed and effective technique for NMR exploration of protein structure (Crespi et al., 1968; Markley et al., 1968). This approach allowed more facile assignment of specific resonances and generated the potential to follow the spectroscopic behavior of protons for a specific amino acid sidechain over a broad range of conditions. The primary method for labeling at that time involved growth of microorganisms (generally bacteria or algae) in D2O, followed by isolation of the deuteratedamino acids from a cellular protein hydrolysate. The amino acids isolated were, therefore, completely deuterated. Selective deuteration of a target protein was achieved by growing the producing organism on a mixture of completely deuterated and selected protonated amino acids under conditions that minimized metabolic interconversion of the amino acids. In one-dimensional spectra, aromatic amino acid resonances occur well downfield of the aliphatic resonances, and this region can therefore be examined somewhat independently by utilizing a single protonated aromatic amino acid to simplify the spectrum of the protein. However, the multiple spectral lines generated by aromatic amino acids can be complex and overlapping, precluding unequivocal interpretation. To address this complication, chemical methods were developed to both completely and selectively deuterate side chains of the aromatic amino acids, thereby avoiding the costly necessity of growing large volumes of microorganisms in D2O and subsequent tedious isolation procedures. In addition, selective deuteration of the amino acids simplified the resonance patterns and thereby facilitated assignment and interpretation of spectra. The methods employed were based on exchange phenomena reported in the literature and generated large quantities of material for use in growth of microorganisms for subsequent isolation of selectively labeled protein (Matthews et al., 1977a). The target protein for incorporation of the selectively deuterated aromatic amino acids generated by these chemical methods was the lactose repressor protein from Escherichia coli, and greatly simplified spectra of this 150,000 D protein were produced by this approach.

Amino Acid Metabolism in Plants. III. Purification and Some Properties of a Multispecific Aminotransferase Isolated from Bushbean Seedlings (Phaseolus vulgaris L.)

1972 ◽  
Vol 50 (7) ◽  
pp. 813-829 ◽  
Author(s):  
J. C. Forest ◽  
F. Wightman
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Aspartic Acid ◽  
Aspartate Aminotransferase ◽  
Soluble Protein ◽  
Specific Activity ◽  
Aromatic Amino Acids ◽  
Total Activity ◽  
Aminotransferase Activity ◽  
Aromatic Aminotransferase

The development of aromatic aminotransferase activity was examined in cotyledons, roots, and shoots of bushbean seedlings growing under light or dark conditions for the first 2 weeks after germination. All three aromatic amino acid – α-ketoglutarate aminotransferase activities were found to have similar patterns of development in comparable organs grown under the two environmental conditions, and the changes in levels of activity appeared unrelated to variations in the endogenous amounts of free aromatic amino acids in the organs of these seedlings. The highest total activity for all three transamination reactions was found in the shoots of light-grown seedlings after 14 days, whereas the aminotransferases showing highest specific activity were found in roots of both kinds of seedlings after 8 days of growth. The intracellular distribution of the three aromatic aminotransferase activities and of aspartate aminotransferase activity was investigated by differential centrifugation of root homogenates. Only a total of 10% of these two activities was found in the two particulate fractions; the soluble protein in the final supernatant fraction accounted for almost 90% of the total aromatic and aspartate aminotransferase activities.The aromatic aminotransferase in the soluble protein fraction from seedling roots was purified about 600-fold by pH precipitation, ammonium sulfate fractionation, and Sephadex chromatography, and the recovery obtained was 30–35% based on total activity. It was observed that the specific activity for aspartate–α-ketoglutarate aminotransferase increased proportionally to the increase in aromatic aminotransferase activities during the different steps of purification. Gel electrophoresis of the purified fraction revealed only one protein band which corresponded to the product-specific stained band for the three aromatic aminotransferase activities assayed on other gels. The molecular weight of the purified aminotransferase was found to be about 128 000 daltons and its Stokes radius was calculated to be 43 ± 3 Å. The pH optima for the three aromatic aminotransferase activities and for aspartate aminotransferase activity were all found to be 8.5. The purified enzyme showed no specific requirement for pyridoxal phosphate and an examination of its amino acid substrate specificity revealed that it was able to catalyze transamination of L-aspartic acid, L-phenylalanine, L-tyrosine, and L-tryptophan when α-ketoglutarate was provided as amino group acceptor. The enzyme was also found to catalyze transamination of L-glutamic acid when oxaloacetate was used as amino group acceptor, but neither pyruvate nor glyoxylate were utilized as amino acceptors for transamination of any of the amino acids examined. The enzyme was found to catalyze transamination of aspartic acid with much greater velocity than its rate of reaction with any of the three aromatic amino acids, and the inclusion of aspartic acid in a reaction medium at equimolar concentration with any one of the three aromatic amino acids resulted in strong inhibition of the aromatic aminotransferase activity of the enzyme. All the evidence indicates that the soluble protein fraction purified from bushbean roots contained only one aminotransferase which was able to catalyze the transamination of five L-amino acids. The demonstration of the substrate multispeciftcity of this pure enzyme represents the first evidence for a multispecific aminotransferase in plants.

Escherichia coli aromatic amino acid aminotransferase: Characterization and comparison with aspartate aminotransferase

Biochemistry ◽  
1993 ◽  
Vol 32 (45) ◽  
pp. 12229-12239 ◽  
Author(s):  
Hideyuki Hayashi ◽  
Katsura Inoue ◽  
Toshihito Nagata ◽  
Seiki Kuramitsu ◽  
Hiroyuki Kagamiyama
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Aspartate Aminotransferase ◽  
Aromatic Amino Acid ◽  
Aromatic Amino Acid Aminotransferase

Amino Acid Profile and Aromatic Amino Acid Concentration in Total Parenteral Nutrition: Effect on Growth, Protein Metabolism and Aromatic Amino Acid Metabolism in the Neonatal Piglet

1994 ◽  
Vol 87 (1) ◽  
pp. 75-84 ◽  
Author(s):  
Linda J. Wykes ◽  
James D. House ◽  
Ronald O. Ball ◽  
Paul B. Pencharz
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Parenteral Nutrition ◽  
Human Milk ◽  
Total Parenteral Nutrition ◽  
Aromatic Amino Acid ◽  
Nitrogen Retention ◽  
Aromatic Amino Acids ◽  
The Other ◽  
Constant Infusion

1. The protein and amino acid utilization of two commercially available amino acid solutions, one egg-patterned (Vamin), the other human-milk-patterned (Vaminolact), were studied in piglets receiving total parenteral nutrition. It was hypothesized that Vaminolact was deficient in total aromatic amino acids, so a third group received a human-milk-patterned amino acid solution with added phenylalanine. 2. The piglets were on total parenteral nutrition for 8 days from day 2 or 3 of life. They all received a total energy intake of 1040 kJ day−1 kg−1 with macro-nutrient intakes of 14.6g of amino acid, 27.4 g of glucose and 9.4 g of fat day−1 kg−1. 3. Nitrogen balances were performed on days 3-8 of total parenteral nutrition. On day 8 a primed constant infusion of (1-14C]-phenylalanine was given to measure phenylalanine flux and fractional conversion to tyrosine. Transamination catabolites of phenylalanine and tyrosine were measured in urine on day 7. 4. The piglets receiving Vaminolact gained significantly less weight (0.86 kg compared with 1.18 kg for Vamin and 1.20 kg for phenylalanine-supplemented Vaminolact; P < 0.02) and nitrogen (1435 mg day−1 kg−1 compared with 1601 mg and 1836 mg day−1 kg−1 for the other groups; P < 0.0001). 5. The piglets receiving Vamin had high plasma phenylalanine levels (2234 μmol/l compared with 156 μmol/l for Vaminolact and 399 μmol for phenylalanine-supplemented Vaminolact; P < 0.0001). Those receiving Vamin also had an elevated excretion of phenylalanine transamination metabolites and low plasma lysine levels. Phenylalanine flux was highest in the Vamin group, intermediate in the phenylalanine-supplemented Vaminolact group and lowest in the Vaminolact group. 6. We conclude that Vaminolact is limiting in aromatic amino acids and that the addition of phenylalanine to the level in Vamin significantly improves growth and nitrogen retention; however, increasing the phenylalanine content of total parenteral nutrition is not the most metabolically suitable way to provide aromatic amino acids in neonatal total parenteral nutrition.

Sign in / Sign up

Export Citation Format

Share Document