Publications

• Tyrosine aminotransferase: biochemical and structural properties and molecular dynamics simulations.

  Mehere P., Han Q., Lemkul J.A., Vavricka C.J., Robinson H., Bevan D.R., Li J.

  Tyrosine aminotransferase (TAT) catalyzes the transamination of tyrosine and other aromatic amino acids. The enzyme is thought to play a role in tyrosinemia type II, hepatitis and hepatic carcinoma recovery. The objective of this study is to investigate its biochemical and structural characteristi ... >> More

  Tyrosine aminotransferase (TAT) catalyzes the transamination of tyrosine and other aromatic amino acids. The enzyme is thought to play a role in tyrosinemia type II, hepatitis and hepatic carcinoma recovery. The objective of this study is to investigate its biochemical and structural characteristics and substrate specificity in order to provide insight regarding its involvement in these diseases. Mouse TAT (mTAT) was cloned from a mouse cDNA library, and its recombinant protein was produced using Escherichia coli cells and purified using various chromatographic techniques. The recombinant mTAT is able to catalyze the transamination of tyrosine using α-ketoglutaric acid as an amino group acceptor at neutral pH. The enzyme also can use glutamate and phenylalanine as amino group donors and p-hydroxy-phenylpyruvate, phenylpyruvate and alpha-ketocaproic acid as amino group acceptors. Through macromolecular crystallography we have determined the mTAT crystal structure at 2.9 Å resolution. The crystal structure revealed the interaction between the pyridoxal-5'-phosphate cofactor and the enzyme, as well as the formation of a disulphide bond. The detection of disulphide bond provides some rational explanation regarding previously observed TAT inactivation under oxidative conditions and reactivation of the inactive TAT in the presence of a reducing agent. Molecular dynamics simulations using the crystal structures of Trypanosoma cruzi TAT and human TAT provided further insight regarding the substrate-enzyme interactions and substrate specificity. The biochemical and structural properties of TAT and the binding of its cofactor and the substrate may help in elucidation of the mechanism of TAT inhibition and activation. << Less

  Protein Cell 1:1023-1032(2010) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Characterization of aromatic aminotransferases from the hyperthermophilic archaeon Thermococcus litoralis.

  Andreotti G., Cubellis M.V., Nitti G., Sannia G., Mai X., Marino G., Adams M.W.

  The hyperthermophilic archaeon (formerly archaebacterium) Thermococcus litoralis grows at temperatures up to 98 degrees C using peptides and proteins as the sole sources of carbon and nitrogen. Cell-free extracts of the organism contained two distinct types of aromatic aminotransferases (EC 2.6.1. ... >> More

  The hyperthermophilic archaeon (formerly archaebacterium) Thermococcus litoralis grows at temperatures up to 98 degrees C using peptides and proteins as the sole sources of carbon and nitrogen. Cell-free extracts of the organism contained two distinct types of aromatic aminotransferases (EC 2.6.1.57) which were separated and purified to electrophoretic homogeneity. Both enzymes are homodimers with subunit masses of approximately 47 kDa and 45 kDa. Using 2-oxoglutarate as the amino acceptor, each catalyzed the pyridoxal-5'-phosphate-dependent transamination of the three aromatic amino acids but showed virtually no activity towards aspartic acid, alanine, valine or isoleucine. From the determination of Km and kcat values using 2-oxoglutarate, phenylalanine, tyrosine and tryptophan as substrates, both enzymes were shown to be highly efficient at transaminating phenylalanine (kcat/Km approximately 400 s-1 mM-1); the 47-kDa enzyme showed more activity towards tyrosine and tryptophan compared to the 45-kDa one. Kinetic analyses indicated a two-step mechanism with a pyridoxamine intermediate. Both enzymes were virtually inactive at 30 degrees C and exhibited maximal activity between 95-100 degrees C. They showed no N-terminal sequence similarity with each other (approximately 30 residues), nor with the complete amino acid sequences of aromatic aminotransferases from Escherichia coli and rat liver. The catalytic properties of the two enzymes are distinct from bacterial aminotransferases, which have broad substrate specificities, but are analogous to two aromatic aminotransferases which play a biosynthetic role in a methanogenic archaeon. In contrast, it is proposed that one or both play a catabolic role in proteolytic T. litoralis in which they generate glutamate and an arylpyruvate. These serve as substrates for glutamate dehydrogenase and indolepyruvate ferredoxin oxidoreductase in a novel pathway for the utilization of aromatic amino acids. << Less

  Eur. J. Biochem. 220:543-549(1994) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

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

  Heilbronn J., Wilson J., Berger B.J.

  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 ... >> More

  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. << Less

  J. Bacteriol. 181:1739-1747(1999) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.