title: Characterization of a thiamin diphosphate-dependent phenylpyruvate decarboxylase from Saccharomyces cerevisiae.
abstract: The product of the ARO10 gene from Saccharomyces cerevisiae was initially identified as a thiamine diphosphate-dependent phenylpyruvate decarboxylase with a broad substrate specificity. It was suggested that the enzyme could be responsible for the catabolism of aromatic and branched-chain amino acids, as well as methionine. In the present study, we report the overexpression of the ARO10 gene product in Escherichia coli and the first detailed in vitro characterization of this enzyme. The enzyme is shown to be an efficient aromatic 2-keto acid decarboxylase, consistent with it playing a major in vivo role in phenylalanine, tryptophan and possibly also tyrosine catabolism. However, its substrate spectrum suggests that it is unlikely to play any significant role in the catabolism of the branched-chain amino acids or of methionine. A homology model was used to identify residues likely to be involved in substrate specificity. Site-directed mutagenesis on those residues confirmed previous studies indicating that mutation of single residues is unlikely to produce the immediate conversion of an aromatic into an aliphatic 2-keto acid decarboxylase. In addition, the enzyme was compared with the phenylpyruvate decarboxylase from Azospirillum brasilense and the indolepyruvate decarboxylase from Enterobacter cloacae. We show that the properties of the two phenylpyruvate decarboxylases are similar in some respects yet quite different in others, and that the properties of both are distinct from those of the indolepyruvate decarboxylase. Finally, we demonstrate that it is unlikely that replacement of a glutamic acid by leucine leads to discrimination between phenylpyruvate and indolepyruvate, although, in this case, it did lead to unexpected allosteric activation.
authors.affiliation: Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis, IN 46202, USA.
authors.forename: Malea M
authors.initials: MM
authors.lastname: Kneen
authors.forename: Razvan
authors.initials: R
authors.lastname: Stan
authors.forename: Alejandra
authors.initials: A
authors.lastname: Yep
authors.forename: Ryan P
authors.initials: RP
authors.lastname: Tyler
authors.forename: Choedchai
authors.initials: C
authors.lastname: Saehuan
authors.forename: Michael J
authors.initials: MJ
authors.lastname: McLeish
mesh_terms: D002262:Carboxy-Lyases / Q000235:genetics / Q000378:metabolism*; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D013058:Mass Spectrometry; D008958:Models, Molecular; D016296:Mutagenesis; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity; D013835:Thiamine Pyrophosphate / Q000378:metabolism*
chemical_list: D002262:Carboxy-Lyases; C109058:phenylpyruvate decarboxylase; D013835:Thiamine Pyrophosphate