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首页> 外文期刊>The FEBS journal >Characterization of a thiamin diphosphate-dependent phenylpyruvate decarboxylase from Saccharomyces cerevisiae
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Characterization of a thiamin diphosphate-dependent phenylpyruvate decarboxylase from Saccharomyces cerevisiae

机译：酿酒酵母中硫胺素二磷酸依赖性苯丙酮酸脱羧酶的表征

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摘要

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
机译：来自酿酒酵母的ARO10基因的产物最初被鉴定为具有广泛底物特异性的硫胺二磷酸依赖性苯丙酮酸脱羧酶。提示该酶可能是芳香族和支链氨基酸以及蛋氨酸的分解代谢的原因。在本研究中，我们报告了ARO10基因产物在大肠杆菌中的过表达以及该酶的首次详细体外表征。该酶显示是一种有效的芳香族2-酮酸脱羧酶，与其在苯丙氨酸，色氨酸以及酪氨酸分解代谢中起主要的体内作用相一致。但是，其底物光谱表明，它不太可能在支链氨基酸或蛋氨酸的分解代谢中发挥重要作用。同源性模型用于鉴定可能与底物特异性有关的残基。这些残基的定点诱变证实了先前的研究，表明单个残基的突变不太可能使芳香族立即转化为脂族2-酮酸脱羧酶。另外，将该酶与来自巴西固氮螺菌的苯基丙酮酸脱羧酶和来自阴沟肠杆菌的吲哚丙酮酸脱羧酶进行了比较。我们表明，这两种丙酮丙酮酸脱羧酶的性质在某些方面相似，而在另一些方面则完全不同，并且两者的性质都与吲哚丙酮酸脱羧酶的性质不同。最后，我们证明亮氨酸替代谷氨酸不太可能导致苯丙酮酸和吲哚丙酮酸之间的区别，尽管在这种情况下确实导致了意外的变构活化。

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