Purification and partial structural and kinetic characterization of tyrosine aminotransferase from epimastigotes of Trypanosoma cruzi.
ABSTRACT: Tyrosine aminotransferase was purified to homogeneity from epimastigotes of Trypanosoma cruzi by a method involving chromatography on DEAE-cellulose, gel filtration on Sephacryl S-200 and chromatography on Mono Q in an f.p.l.c. system. The purified enzyme showed a single band in SDS/PAGE, with an apparent molecular mass of 45 kDa. Since the apparent molecular mass of the native enzyme, determined by gel filtration, is 91 kDa, the native enzyme is a dimer of similar subunits. The amino-acid composition was determined, as well as the sequences of three internal peptides obtained by CNBr cleavage at Met residues. Both criteria suggest considerable similarity with the tyrosine aminotransferases from rat and from human liver. The enzyme contains nine 1/2 Cys residues, three free and the others forming three disulphide bridges. The enzyme is not N-glycosylated. The isoelectric point is 4.6-4.8. The optimal pH for the reaction of the enzyme with tyrosine as a substrate is 7.0. The apparent Km values for tyrosine, phenylalanine and tryptophan, with pyruvate as a co-substrate, were 6.8, 17.9 and 21.4 mM, respectively, whereas those for pyruvate, alpha-oxoglutarate and oxaloacetate, with tyrosine as a substrate, were 0.5, 38 and 16 mM respectively. The purified tyrosine aminotransferase acts as an alanine aminotransferase as well and the activity seems to reside in the same enzyme molecule. The results suggest that the enzyme is a general aromatic-amino-acid transaminase, with high sequence similarity to tyrosine aminotransferases from rat and human liver.
Project description:1. Histidine-pyruvate aminotransferase (isoenzyme 1) was purified to homogeneity from the mitochondrial and supernatant fractions of rat liver, as judged by polyacrylamide-gel electrophoresis and isolectric focusing. Both enzyme preparations were remarkably similar in physical and enzymic properties. Isoenzyme 1 had pI8.0 and a pH optimum of 9.0. The enzyme was active with pyruvate as amino acceptor but not with 2-oxoglutarate, and utilized various aromatic amino acids as amino donors in the following order of activity: phenylalanine greater than tyrosine greater than histidine. Very little activity was found with tryptophan and 5-hydroxytryptophan. The apparent Km values were about 2.6mM for histidine and 2.7 mM for phenylalanine. Km values for pyruvate were about 5.2mM with phenylalanine as amino donor and 1.1mM with histidine. The aminotransferase activity of the enzyme towards phenylalanine was inhibited by the addition of histidine. The mol.wt. determined by gel filtration and sucrose-density-gradient centrifugation was approx. 70000. The mitochondrial and supernatant isoenzyme 1 activities increased approximately 25-fold and 3.2-fold respectively in rats repeatedly injected with glucagon for 2 days. 2. An additional histidine-pyruvate aminotransferase (isoenzyme 2) was partially purified from both the mitochondrial and supernatant fractions of rat liver. Nearly identical properties were observed with both preparations. Isoenzyme 2 had pI5.2 and a pH optimum of 9.3. The enzyme was specific for pyruvate and did not function with 2-oxoglutarate. The order of effectiveness of amino donors was tyrosine = phenylalanine greater than histidine greater than tryptophan greater than 5-hydroxytryptophan. The apparent Km values for histidine and phenylalanine were about 0.51 and 1.8 mM respectively. Km values for pyruvate were about 3.5mM with phenylalanine and 4.7mM with histidine as amino donors. Histidine inhibited phenylalanine aminotransferase activity of the enzyme. Gel filtration and sucrose-density-gradient centrifugation yielded a mol.wt. of approx. 90000. Neither the mitochondrial nor the supernatant isoenzyme 2 activity was elevated by glucagon injection.
Project description: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.
Project description: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.
Project description:The organ distribution of rat histidine-pyruvate aminotransferase isoenzymes 1 and 2 was examined by using an isoelectric-focusing technique. Isoenzyme 1 (pI8.0) is present only in the liver and its activity is increased by the injection of glucagon, whereas isoenzyme 2 (pI5.2) is distributed in all tissues (liver, kidney, brain and heart) tested, and is not affected by glucagon injection. Isoenzyme 2 of the liver, kidney, brain and heart was purified by the same procedure and characterized. Isoenzyme 2 preparations from these four tissues were nearly identical in physical and enzymic properties. These properties differed from those previously found for the highly purified isoenzyme 1 preparation of rat liver. Isoenzyme 2 was active with pyruvate but not with 2-oxoglutarate as amino acceptor. Amino donors were effective in the following order of activity: tyrosine greater than histidine greater than phenylalanine greater than kynurenine greater than tryptophan. Very little activity was found with 5-hydroxytryptophan. The apparent Km for histidine was about 0.45 mM. The Km for pyruvate was about 4.5 mM with histidine as amino donor. The amino-transferase activities of isoenzyme 2 towards phenylalanine and tyrosine were inhibited by histidine. The ratio of aminotransferase activities towards these three amino acids was constant through gel filtration, electrophoresis, isoelectric focusing and sucrose-density-gradient centrifugation of the purified isoenzyme 2 preparations. These results suggest that these three activities are properties of the same enzyme protein. Sephadex G-150 gel filtration and sucrose-density-gradient centrifugation yielded mol.wts. of approx. 95000 and 92000 respectively. The pH optimum was between 9.0 and 9.3.
Project description:Kynurenine-glyoxylate aminotransferase, alanine-glyoxylate aminotransferase and serine-pyruvate aminotransferase were co-purified and crystallized as yellow cubes from human liver particulate fraction. The crystalline enzyme was homogeneous by the criteria of electrophoresis, isoelectric focusing, gel filtration, sucrose-density-gradient centrifugation and analytical ultracentrifugation. The molecular weight of the enzyme was calculated as approx. 90000, 89000 and 99000 by the use of gel filtration, analytical ultracentrifugation and sucrose-density-gradient centrifugation respectively, with two identical subunits. The enzyme has a s(20,w) value of 5.23S, an isoelectric point of 8.3 and a pH optimum between 9.0 and 9.5. The enzyme solution showed absorption maxima at 280 and 420nm. The enzyme catalysed transamination between several l-amino acids and pyruvate or glyoxylate. The order of effectiveness of amino acids was alanine>serine>glutamine>glutamate>methionine>kynurenine = phenylalanine = asparagine>valine>histidine>lysine>leucine>isoleucine>arginine>tyrosine = threonine>aspartate, with glyoxylate as amino acceptor. The enzyme was active with glyoxylate, oxaloacetate, hydroxypyruvate, pyruvate, 4-methylthio-2-oxobutyrate and 2-oxobutyrate, but showed little activity with phenylpyruvate, 2-oxoglutarate and 2-oxoadipate, with kynurenine as amino donor. Kynurenine-glyoxylate aminotransferase activity was competitively inhibited by the addition of l-alanine or l-serine. From these results we conclude that kynurenine-glyoxylate aminotransferase, alanine-glyoxylate aminotransferase and serine-pyruvate aminotransferase activities of human liver are catalysed by a single protein. Kinetic parameters for the kynurenine-glyoxylate aminotransferase, alanine-glyoxylate aminotransferase, serine-pyruvate aminotransferase and alanine-hydroxypyruvate aminotransferase reactions of the enzyme are presented.
Project description:Tyrosine aminotransferase (TAT) is an aminotransferase with broad substrate specificity that catalyzes the transamination of aromatic amino acids in Leishmania donovani and plays a crucial role in the survival and pathogenicity of the parasite. In this study, we have biochemically characterized tyrosine aminotransferase from Leishmania donovani using in vitro and in silico techniques. Leishmania donovani tyrosine aminotransferase (LdTAT) was cloned into the pET28a(+) vector and expressed in the BL21 strain of Escherichia coli. The Ni-NTA-purified protein was then characterized biochemically, and its various kinetic parameters were investigated. The apparent Km value for the tyrosine-pyruvate pair was determined to be 3.5 ± 0.9 mm, and Vmax was analyzed to be at 11.7 ± 1.5 ?m·min.?g-1 . LdTAT was found to exhibit maximum activity at 50 °C and at a pH of 8.0. Cofactor identification for LdTAT showed that pyridoxal-5-phosphate (PLP) binds with a Km value of 23.59 ± 3.99 ?m and that the phosphate group is vital for the activity of the enzyme. Sequence analysis revealed that S151, Y256, K286, and P291 are conserved residues and form hydrogen bonds with PLP. Urea-based denaturation studies revealed a biphasic folding mechanism involving N?X?D states. Molecular dynamic simulations of modeled LdTAT at various conditions were performed to understand enzyme behavior and interactions at the molecular level. The biochemical and structural divergence between host and parasite TAT suggests the LdTAT has evolved to utilize pyruvate rather than ?-ketoglutarate as co-substrate. Furthermore, our data suggest that LdTAT may be a potential drug target due to its divergence in structure and substrate specificity from the host.
Project description:The aminotransferase gene family in the model plant Arabidopsis thaliana consists of 44 genes, eight of which are suggested to be alanine aminotransferases. One of the putative alanine aminotransferases genes, At3g08860, was attributed the function of alanine:glyoxylate aminotransferase/?-alanine:pyruvate aminotransferase based on the analysis of gene expression networks and homology to other ?-alanine aminotransferases in plants. It was earlier demonstrated that At3g08860 is specifically upregulated in response to osmotic stress, but not other stresses (?-alanine is an osmoprotectant in plants). Furthermore, it was shown that the expression of At3g08860 is highly coordinated with the genes of the uracil degradation pathway leading to the non-proteinogenic amino acid ?-alanine. These evidence were suggestive of the involvement of At3g08860 in ?-alanine metabolism. However, direct experimental evidence for the function of At3g08860 was lacking, and therefore, the goal of this study was to elucidate the function of the uncharacterized aminotransferase annotated by the locus tag At3g08860. The cDNA of At3g08860 was demonstrated to functionally complement two E. coli mutants auxotrophic for the amino acids, L-alanine (proteinogenic) and ?-alanine (non-proteinogenic). Enzyme activity using purified recombinant At3g08860 further demonstrated that the enzyme is endowed with L-alanine:glyoxylate aminotransferase activity.
Project description:1. Kynurenine-2-oxoglutarate aminotransferase (isoenzyme 1) was purified to homogeneity from the liver, brain and small intestine of rats by the same procedure. The three enzyme preparations had nearly identical pH optima, substrate specificities and molecular weights. Isoenzyme 1 was active with 2-oxoglutarate but not with pyruvate as amino acceptor, and utilized a wide range of amino acids as amino donors. Amino acids were effective in the following order to activity: L-aspartate greater than L-tyrosine greater than L-phenylalanine greater than L-tryptophan greater than 5-hydroxy-L-tryptophan greater than L-kynurenine. The molecular weight was approximately 88 000 as determined by sucrose-density-gradient centrifugation. The pH optimum was between 8.0 and 8.5. On the basis of substrate specificity, substrate inhibition, subcellular distribution and polyacrylamide-disc-gel electrophoresis, it is suggested that liver, brain and small intestinal kynurenine-2-oxoglutarate aminotransferase (isoenzyme 1) is identical with mitochondrial tyrosine-2-oxoglutarate aminotransferase and also with mitochondrial aspartate-2-oxoglutarate aminotransferase. 2. An additional kynurenine-2-oxoglutarate aminotransferase (isoenzyme 2) was purified from the liver. This enzyme was specific for 2-oxoglutarate and L-kynurenine. Sucrose-density-gradient centrifugation gave a molecular weight of approximately 100 000. The pH optimum was between 6.0 and 6.5. This enzyme was not detected in the brain or small intestine.
Project description:The crystal structure of tyrosine aminotransferase (TAT) from the parasitic protozoan Trypanosoma cruzi, which belongs to the aminotransferase subfamily Igamma, has been determined at 2.5 A resolution with the R-value R = 15.1%. T. cruzi TAT shares less than 15% sequence identity with aminotransferases of subfamily Ialpha but shows only two larger topological differences to the aspartate aminotransferases (AspATs). First, TAT contains a loop protruding from the enzyme surface in the larger cofactor-binding domain, where the AspATs have a kinked alpha-helix. Second, in the smaller substrate-binding domain, TAT has a four-stranded antiparallel beta-sheet instead of the two-stranded beta-sheet in the AspATs. The position of the aromatic ring of the pyridoxal-5'-phosphate cofactor is very similar to the AspATs but the phosphate group, in contrast, is closer to the substrate-binding site with one of its oxygen atoms pointing toward the substrate. Differences in substrate specificities of T. cruzi TAT and subfamily Ialpha aminotransferases can be attributed by modeling of substrate complexes mainly to this different position of the cofactor-phosphate group. Absence of the arginine, which in the AspATs fixes the substrate side-chain carboxylate group by a salt bridge, contributes to the inability of T. cruzi TAT to transaminate acidic amino acids. The preference of TAT for tyrosine is probably related to the ability of Asn17 in TAT to form a hydrogen bond to the tyrosine side-chain hydroxyl group.
Project description:Alanine aminotransferase (AlaAT) was purified from cell extracts of the hyperthermophilic archaeon Pyrococcus furiosus by multistep chromatography. The enzyme has an apparent molecular mass of 93.5 kDa, as estimated by gel filtration, and consists of two identical subunits of 46 kDa, as deduced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the gene sequence. The AlaAT displayed a broader substrate specificity than AlaATs from eukaryal sources and exhibited significant activity with alanine, glutamate, and aspartate with either 2-oxoglutarate or pyruvate as the amino acceptor. Optimal activity was found in the pH range of 6. 5 to 7.8 and at a temperature of over 95 degrees C. The N-terminal amino acid sequence of the purified AlaAT was determined and enabled the identification of the gene encoding AlaAT (aat) in the P. furiosus genome database. The gene was expressed in Escherichia coli, and the recombinant enzyme was purified. The pH and temperature dependence, molecular mass, and kinetic parameters of the recombinant were indistinguishable from those of the native enzyme from P. furiosus. The k(cat)/K(m) values for alanine and pyruvate formation were 41 and 33 s(-1) mM(-1), respectively, suggesting that the enzyme is not biased toward either the formation of pyruvate, or alanine. Northern analysis identified a single 1.2-kb transcript for the aat gene. In addition, both the aat and gdh (encoding the glutamate dehydrogenase) transcripts appear to be coregulated at the transcriptional level, because the expression of both genes was induced when the cells were grown on pyruvate. The coordinated control found for the aat and gdh genes is in good agreement with these enzymes acting in a concerted manner to form an electron sink in P. furiosus.