UNIPROT:P17174 - FACTA Search

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Query: UNIPROT:P17174 (aspartate aminotransferase)
14,872 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Tyrosine phenol-lyase (TPL), which catalyzes the beta-elimination reaction of L-tyrosine, and aspartate aminotransferase (AspAT), which catalyzes the reversible transfer of an amino group from dicarboxylic amino acids to oxo acids, both belong to the alpha-family of vitamin B6-dependent enzymes. To switch the substrate specificity of TPL from L-tyrosine to dicarboxylic amino acids, two amino acid residues of AspAT, thought to be important for the recognition of dicarboxylic substrates, were grafted into the active site of TPL. Homology modeling and molecular dynamics identified Val-283 in TPL to match Arg-292 in AspAT, which binds the distal carboxylate group of substrates and is conserved among all known AspATs. Arg-100 in TPL was found to correspond to Thr-109 in AspAT, which interacts with the phosphate group of the coenzyme. The double mutation R100T/V283R of TPL increased the beta-elimination activity toward dicarboxylic amino acids at least 10(4)-fold. Dicarboxylic amino acids (L-aspartate, L-glutamate, and L-2-aminoadipate) were degraded to pyruvate, ammonia, and the respective monocarboxylic acids, e.g. formate in the case of L-aspartate. The activity toward L-aspartate (kcat = 0.21 s-1) was two times higher than that toward L-tyrosine. beta-Elimination and transamination as a minor side reaction (kcat = 0.001 s-1) were the only reactions observed. Thus, TPL R100T/V283R accepts dicarboxylic amino acids as substrates without significant change in its reaction specificity. Dicarboxylic amino acid beta-lyase is an enzyme not found in nature.
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PMID:Conversion of tyrosine phenol-lyase to dicarboxylic amino acid beta-lyase, an enzyme not found in nature. 988 May 2

Chronic occupational exposure to organophosphorus and carbamate-type pesticides significantly inhibits acetylcholinesterase activity and causes morbidity. This study on mice was designed to evaluate their amino profile and to identify signs of hepatic dysfunction following their chronic exposure to mixtures of organophosphorus pesticides. Laboratory mice were exposed to a formulated mixture of the six organophosphorus pesticides (Dimethoate, Chlorpyrifos, Profenofos, Pirimiphos methyl, Triazophos and Dimethoate) most commonly used in agriculture in this region of the Middle East. Doses (10% of LD50 of the mixture) were given once a week by gavage in corn oil for 7 weeks; the control group was given only corn oil. At the end of the exposure period, mice were culled and blood samples were collected to determine erythrocyte acetylcholinesterase activity, biochemical markers of liver function and concentrations of serum amino acids. Erythrocyte acetylcholinesterase activity and total serum proteins decreased significantly in the exposed group. Serum concentrations of alanine aminotransferase and aspartate aminotransferase, alanine, glutamic acid, glycine, isoleucine, leucine, methionine, ornithine, proline, serine, threonine and valine were significantly increased in the exposed mice, while serum levels of cystine were decreased significantly. There were also non-significant increases in serum alkaline phosphatase, gama-glutamyl transpeptidase and some of the other amino acids. Chronic exposure to mixtures of organophosphorus pesticides is associated with decreased acetylcholinesterase activity, hepatic dysfunction and disturbance of amino acids profile. Biochemical indices of hepatocellular injury and disturbed amino acid metabolism may be of value as markers of chronic exposure to such pesticides.
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PMID:Hepatic injury and disturbed amino acid metabolism in mice following prolonged exposure to organophosphorus pesticides. 1002 66

The genes encoding aspartate kinase (ask), homoserine dehydrogenase (hom), homoserine kinase (thrB) and threonine synthase (thrC) from the obligate methylotroph Methylobacillus flagellatus were cloned. In maxicells hom and thrC directed synthesis of 51 and 48 kDa polypeptides, respectively. The hom, thrB and thrC genes and adjacent DNA areas were sequenced. Of the threonine biosynthesis genes, only hom and thrC were tightly linked in the order hom-thrC. The gene for thymidylate synthase (thyA) followed thrC and the gene for aspartate aminotransferase (aspC) preceded hom. All four genes (aspC-hom-thrC-thyA) were transcribed in the same direction. mRNA analysis indicated that hom-thrC are apparently transcribed in one 7.5 kb transcript in M. flagellatus. Promoter analysis showed the presence of a functional promoter between aspC and hom. No functional promoter was found to be associated with the DNA stretch between hom and thrC. The thrB gene encoded an unusual type of homoserine kinase and was not linked to other threonine biosynthesis genes.
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PMID:Organization of threonine biosynthesis genes from the obligate methylotroph Methylobacillus flagellatus. 1058 37

Aromatic amino acid aminotransferase (ArATPh), which has a melting temperature of 120 degrees C, is one of the most thermostable aminotransferases yet to be discovered. The crystal structure of this aminotransferase from the hyperthermophilic archaeon Pyrococcus horikoshii was determined to a resolution of 2.1 A. ArATPh has a homodimer structure in which each subunit is composed of two domains, in a manner similar to other well characterized aminotransferases. By the least square fit after superposing on a mesophilic ArAT, the ArATPh molecule exhibits a large deviation of the main chain coordinates, three shortened alpha-helices, an elongated loop connecting two domains, and a long loop transformed from an alpha-helix, which are all factors that are likely to contribute to its hyperthermostability. The pyridine ring of the cofactor pyridoxal 5'-phosphate covalently binding to Lys(233) is stacked parallel to F121 on one side and interacts with the geminal dimethyl-CH/pi groups of Val(201) on the other side. This tight stacking against the pyridine ring probably contributes to the hyperthermostability of ArATPh. Compared with other ArATs, ArATPh has a novel substrate specificity, the order of preference being Tyr > Phe > Glu > Trp > His>> Met > Leu > Asp > Asn. Its relatively weak activity against Asp is due to lack of an arginine residue corresponding to Arg(292)* (where the asterisk indicates that this is a residues supplied by the other subunit of the dimer) in pig cytosolic aspartate aminotransferase. The enzyme recognizes the aromatic substrate by hydrophobic interaction with aromatic rings (Phe(121) and Tyr(59)*) and probably recognizes acidic substrates by a hydrophilic interaction involving a hydrogen bond network with Thr(264)*.
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PMID:The molecular structure of hyperthermostable aromatic aminotransferase with novel substrate specificity from Pyrococcus horikoshii. 1067 23

The 3-D structural information is a prerequisite for a rational ligand design. In the absence of experimental data, model building on the basis of a known 3-D structure of a homologous protein is at present the only reliable method to obtain structural information. A homology model building study of the pyridoxal 5'-phosphate (PLP)-dependent histidine decarboxylase from Morganella morganii (HDC-MM) has been carried out based on the crystal structure of the aspartate aminotransferase from Escherichia coli (AAT-EC). The primary sequences of AAT-EC and HDC-MM were aligned by automated alignment procedure. A 3-D model of HDC-MM was constructed by copying the coordinates of the residues from the crystal structure of AAT-EC into the corresponding residues in HDC-MM. After energy-minimization of the resulting 3-D model of HDC-MM, possible active site residues were identified by fitting the substrate (l-histidine) into the proposed active-site. In our model, several residues, which have an important role in the AAT-EC active-site, are located in positions spatially identical to those in AAT-EC structure. The back-bone of the modelled active site pocket is constructed by residues; Gly-92, Gly-93, Thr-93, Ser-115, Asp-200, Ala-202, Ser-229 and Lys-232 together with residues Asn-8, His-119, Thr-171, His-198, Leu-203, His-231, Ser-236 and Ile-238. In the ligand binding site, it appears that the HDC-MM model will position l-histidine (substrate) in the area consisting of the residues; Glu-29, Ser-30, Leu-38, His-231 and Lys-232. The nitrogen atom of the imidazole ring (N2) of the substrate is predicted to interact with the carboxylate group of Ser-30. The alpha-carboxylate of histidine points toward the Lys-232 to have electrostatic interaction with its side chain nitrogen atom (N(Z)). In conclusion, this combination of sequence and 3-D structural homology between AAT-EC and HDC-MM model could provide insight in assigning the probable active site residues.
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PMID:Homology-based molecular modelling of PLP-dependent histidine decarboxylase from Mmorganella morganii. 1090 9

2-Amino-3-ketobutyrate CoA ligase (KBL, EC 2.3.1.29) is a pyridoxal phosphate (PLP) dependent enzyme, which catalyzes the second reaction step on the main metabolic degradation pathway for threonine. It acts in concert with threonine dehydrogenase and converts 2-amino-3-ketobutyrate, the product of threonine dehydrogenation by the latter enzyme, with the participation of cofactor CoA, to glycine and acetyl-CoA. The enzyme has been well conserved during evolution, with 54% amino acid sequence identity between the Escherichia coli and human enzymes. We present the three-dimensional structure of E. coli KBL determined at 2.0 A resolution. KBL belongs to the alpha family of PLP-dependent enzymes, for which the prototypic member is aspartate aminotransferase. Its closest structural homologue is E. coli 8-amino-7-oxononanoate synthase. Like many other members of the alpha family, the functional form of KBL is a dimer, and one such dimer is found in the asymmetric unit in the crystal. There are two active sites per dimer, located at the dimer interface. Both monomers contribute side chains to each active/substrate binding site. Electron density maps indicated the presence in the crystal of the Schiff base intermediate of 2-amino-3-ketobutyrate and PLP, an external aldimine, which remained bound to KBL throughout the protein purification procedure. The observed interactions between the aldimine and the side chains in the substrate binding site explain the specificity for the substrate and provide the basis for a detailed proposal of the reaction mechanism of KBL. A putative binding site of the CoA cofactor was assigned, and implications for the cooperation with threonine dehydrogenase were considered.
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PMID:Three-dimensional structure of 2-amino-3-ketobutyrate CoA ligase from Escherichia coli complexed with a PLP-substrate intermediate: inferred reaction mechanism. 1131 37

L-Threonine acetaldehyde-lyase (threonine aldolase, TA) is a pyridoxal-5'-phosphate-dependent (PLP) enzyme that catalyzes conversion of L-threonine or L-allo-threonine to glycine and acetaldehyde in a secondary glycine biosynthetic pathway. X-ray structures of Thermatoga maritima TA have been determined as the apo-enzyme at 1.8 A resolution and bound to substrate L-allo-threonine and product glycine at 1.9 and 2.0 A resolution, respectively. Despite low pairwise sequence identities, TA is a member of aspartate aminotransferase (AATase) fold family of PLP enzymes. The enzyme forms a 222 homotetramer with the PLP cofactor bound via a Schiff-base linkage to Lys199 within a domain interface. The structure reveals bound calcium and chloride ions that appear to contribute to catalysis and oligomerization, respectively. Although L-threonine and L-allo-threonine are substrates for T. maritima TA, enzymatic assays revealed a strong preference for L-allo-threonine. Structures of the external aldimines with substrate/product reveal a pair of histidines that may provide flexibility in substrate recognition. Variation in the threonine binding pocket may explain preferences for L-allo-threonine versus L-threonine among TA family members.
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PMID:X-ray structures of threonine aldolase complexes: structural basis of substrate recognition. 1226 13

Raf-1 protein serine threonine kinase plays an important role in cell survival and proliferation. Antisense inhibition of Raf-1 expression has been shown to enhance the cytotoxic effects of radiation and anticancer drugs. Here we have evaluated the toxicity, pharmacokinetics, and antitumor efficacy of a novel formulation of liposome-entrapped raf antisense oligodeoxyribonucleotide (LErafAON). The LErafAON preparation showed high liposome entrapment efficiency of rafAON (>85%) and stability at room temperature. In CD2F1 mice, administration of LErafAON produced no morbidity/mortality (5-35 mg/kg/dose, i.v., x12). Dose-related elevations in liver enzymes (alanine aminotransferase and aspartate aminotransferase) and histopathological changes in liver were noted in LErafAON and blank liposome groups. No morbidity/mortality and changes in clinical chemistry or histopathology were observed in New Zealand white rabbits (3.75 mg/kg/dose, i.v., x8; 6.5 mg/kg/dose, i.v., x6) or in cynomolgous monkeys (3.75 or 6.25 mg/kg/dose, i.v., x9). Transient decrease in total hemolytic complement activity (approximately 62-74%) and increases in C3a (approximately 3-fold) and Bb levels (approximately 5-12-fold) were observed in LErafAON and blank liposome groups of monkeys. A 30 mg/kg i.v. dose of LErafAON in human prostate tumor (PC-3)-bearing BALB/c athymic mice gave a terminal plasma half-life of 27 h, and intact rafAON could be detected in plasma and in normal and tumor tissues for up to at least 48 h. In monkeys, the terminal plasma half-life of 30.36 +/- 23.87 h was observed at an i.v. dose of 6.25 mg/kg. LErafAON (25 mg/kg/dose, i.v., x10) or ionizing radiation (3.8 Gy/day, x5) treatment of PC-3 tumor-bearing athymic mice led to tumor growth arrest, whereas a combination of LErafAON and ionizing radiation treatments resulted in tumor regression. LErafAON treatment caused inhibition of Raf-1 protein expression in normal and tumor tissues in these mice (>50%, versus controls). These data have formed a basis of the clinical Phase I studies of LErafAON for cancer treatment.
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PMID:Pharmacokinetics, toxicity, and efficacy of ends-modified raf antisense oligodeoxyribonucleotide encapsulated in a novel cationic liposome. 1242 53

Microcystins are usually the predominant cyanotoxins present in both drinking and recreational waters after cyanobacterial blooms. Their classic toxic effect is hepatotoxicity through inhibition of serine/threonine phosphatases. However, recent studies also reported oxidative stress generation and disruption of ion regulation in aquatic organisms after microcystins exposure. In the present study, aqueous extracts of Microcystis aeruginosa were administered to the estuarine crab Chasmagnathus granulatus (Decapoda, Brachyura) by gavage in variable doses (from 34 to 860 microg kg(-1)) and exposure times (6, 12, and 72 h). A control group was exposed to saline solution. Analyzed variables included oxygen consumption, lipid peroxidation (LPO), enzyme activities (glutathione S-transferases or GST; alanine aminotransferase or ALT; aspartate aminotransferase or AST; and lactate dehydrogenase or LDH), glycogen, and microcystins content. Oxygen consumption increased in organisms exposed for 12h to 860 microg kg(-1) of microcystins and a similar result was observed after 72 h at doses equal to or higher than 34 microg kg(-1). LPO levels increased in doses equal to or higher than 34 microg kg(-1) after 72 h. GST and LDH activities increased after 12 h (at a dose of 860 microg kg(-1)), but ALT and AST activities remained unaltered in all experimental conditions. Glycogen content decreased after 72 h exposure at doses equal to or higher than 172 microg kg(-1). After 12h of exposure to 860 microg kg(-1) of microcystins, the concentration found in the hepatopancreas of C. granulatus was 13.17+/-0.56 microg kg(-1). In crabs exposed to doses higher than 172 microg kg(-1) during 72 h this value raised to 32.14+/-4.12 microg kg(-1). The obtained results indicated that microcystins exposure led the tissue to an oxidative stress condition (high LPO levels), at least in part favored by the augment of oxygen consumption, altering the glycogen metabolism. GST responses were only observed in the short-term experiment (12 h) and no effect on classical markers of vertebrate liver damage (ALT and AST) was observed. Although the hepatopancreas from C. granulatus accumulated a relatively low concentration of toxins, it was enough to induce physiological and biochemical disturbances.
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PMID:Biochemical and physiological responses after exposure to microcystins in the crab Chasmagnathus granulatus (Decapoda, Brachyura). 1613 63

The free amino acid concentrations in cotyledons and axes of soybean (Glycine max [L.] Merr. cv. Wells) seedlings were determined by automated single column analysis after germination at 10 and 23 C. After 5 days germination at 10 C, glutamate and aspartate were in high concentration in both cotyledons and axes (38 and 24% of total free amino acids recovered, respectively), whereas the concentrations of their amide derivatives, asparagine and glutamine, were low in cotyledons (4.4%) and high in axes (21%). In contrast, after 5 days germination at 23 C, asparagine and glutamine accounted for 22 and 45% of total free amino acids in cotyledons and axes respectively, and aspartate and glutamate concentrations were low. The activities of glutamine synthetase and asparagine synthetase were considerably lower in tissues from the 10 C treatment than those from the 23 C treatment.Aspartate and glutamate concentrations were nearly equal in all but one sample. Both glutamate oxaloacetate transaminase and glutamate dehydrogenase activities were much higher in axis tissues at 23 C as compared to 10 C. Arrhenius plots of axis glutamate oxaloacetate transaminase and glutamate dehydrogenase activities were biphasic and triphasic, respectively, with energies of activation for both increasing with low temperature. Energies of activation were identical for glutamate oxaloacetate transaminase from 10 and 23 C treatments but much higher for glutamate dehydrogenase from 23 C-treated axes. This indicates a difference in enzyme complement for glutamate dehydrogenase with the two treatments.Hydrolysis of free amino acid sample (basic fraction) aliquots showed large quantities of peptides in 23 C-treated axes at 2 days, while few or no peptides were found in the 10 C treatment. Amino acid residues most prevalent in peptides were aspartate, threonine, serine, glutamate, and glycine.
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PMID:Low Temperature Effects on Soybean (Glycine max [L.] Merr. cv. Wells) Free Amino Acid Pools during Germination. 1666 May 75

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