title: A 1-step microplate method for assessing the substrate range of l-α-amino acid aminotransferase.
abstract: Aminotransferase enzymes catalyse the reversible substitution of a keto group for an amino group. While this reaction is highly stereoselective with respect to the amino group, each enzyme can usually catalyse the turnover of a number of different substrates. As the substrate range cannot be inferred from the sequence, it remains an early bottleneck when selecting an enzyme for a biocatalysis application. We have developed a simple first round characterisation method applicable to the broad range of aminotransferases that accept l-glutamate, the central junction of cellular transamination, as one of the amino donors. The assay is based on l-glutamate detection by its highly specific dehydrogenase enzyme in a coupled assay, ending in the reduction of the 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-tetrazolium-5-carboxanilide (XTT). While products of most tetrazolium salts are water-insoluble, XTT is reduced to a water soluble colored formazan, allowing direct spectrophotometric detection. The reaction is carried out in microplate format using a single endpoint measurement and is thus suitable for automation. The setup was tested with 7 aminotransferase enzymes: Escherichia coli branched chain amino acid aminotransferase, Pseudomonas aeruginosa and Klebsiella pneumoniae aromatic amino acid AT, Bacillus subtilis histidinol-phosphate AT, and Thermus aquaticus aspartate, serine and histidinol-phosphate AT. In addition to 17 of the 20 proteinogenic amino acids, 32 alternative substrates were tested.
authors.affiliation: Research Department of Structural and Molecular Biology, University College London, Gower Street, WC1E 6BT London, UK. martin.bommer@gmx.net
authors.forename: Martin
authors.initials: M
authors.lastname: Bommer
authors.forename: John M
authors.initials: JM
authors.lastname: Ward
mesh_terms: D001219:Aspartate Aminotransferases / Q000378:metabolism; D001412:Bacillus subtilis / Q000201:enzymology; D055162:Biocatalysis; D003124:Colorimetry / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology; D007711:Klebsiella pneumoniae / Q000201:enzymology; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D013237:Stereoisomerism; D013379:Substrate Specificity; D013824:Thermus / Q000201:enzymology; D000637:Transaminases / Q000378:metabolism*
chemical_list: D000637:Transaminases; D001219:Aspartate Aminotransferases; C019798:branched-chain-amino-acid transaminase; C022638:serine-pyruvate aminotransferase; C019729:aromatic amino acid aminotransferase; C053430:histidinol-phosphate aminotransferase