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首页> 外文期刊>Biochimica et Biophysica Acta. Protein Structure and Molecular Enzymology >Recombinant tyrosine aminotransferase from Trypanosoma cruzi: structural characterization and site directed mutagenesis of a broad substrate specificity enzyme
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Recombinant tyrosine aminotransferase from Trypanosoma cruzi: structural characterization and site directed mutagenesis of a broad substrate specificity enzyme

机译：克氏锥虫的重组酪氨酸转氨酶：广泛的底物特异性酶的结构表征和定点诱变

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摘要

The gene encoding tyrosine aminotransferase (TAT, EC 2.6.1.5) from the parasitic protozoan Trypanosoma cruzi was amplified from genomic DNA, cloned into the pET24a expression vector and functionally expressed as a C-terminally His-tagged protein in Escherichia coli BL21(DE3)pLysS. Purified recombinant TAT exhibited identical electrophoretic and enzymatic properties as the authentic enzyme from T. cruzi. Both recombinant and authentic T. cruzi TATs were highly resistant to limited tryptic cleavage and contained no disulfide bonds. Comprehensive analysis of its substrate specificity demonstrated TAT to be a broad substrate aminotransferase, with leucine, methionine as well as tyrosine, phenylalanine, tryptophan and alanine being utilized efficiently as amino donors. Valine, isoleucine and dicarboxylic amino acids served as poor substrates while polar aliphatic amino acids could not be transaminated. TAT also accepted several 2-oxoacids, including 2-oxoisocaproate and 2-oxomethiobutyrate, in addition to pyruvate, oxaloacetate and 2-oxoglutarate. The functionality of the expression system was confirmed by constructing two variants; one (Arg389) being a completely inactive enzyme; the other (Arg283) retaining its full activity, as predicted from the recently solved three-dimensional structure of T. cruzi TAT. Thus, only one of the two strictly conserved arginines which are essential for the enzymatic activity of subfamily Iα aspartate and aromatic aminotransferases is critical for T. cruzi's TAT activity.
机译：从基因组DNA中扩增了来自寄生虫原生动物克氏锥虫的酪氨酸氨基转移酶的编码基因（TAT，EC 2.6.1.5），克隆到pET24a表达载体中，并在大肠杆菌BL21（DE3）中功能性表达为C端His标记蛋白。请纯化的重组TAT表现出与来自克鲁维酵母的真实酶相同的电泳和酶促性质。重组的和真实的克鲁斯锥虫TAT都对有限的胰蛋白酶裂解具有高度抗性，并且不含二硫键。对其底物特异性的全面分析表明，TAT是一种广泛的底物氨基转移酶，亮氨酸，蛋氨酸以及酪氨酸，苯丙氨酸，色氨酸和丙氨酸可有效地用作氨基供体。缬氨酸，异亮氨酸和二羧酸氨基酸是较差的底物，而极性脂族氨基酸则无法转氨。除丙酮酸，草酰乙酸和2-氧戊二酸外，TAT还接受了几种2-氧酸，包括2-氧代己二酸和2-氧代硫代丁酸。表达系统的功能通过构建两个变体得以证实;一种（Arg389）是完全失活的酶;另一个（Arg283）保留了其全部活性，这是根据最近解决的克鲁斯TAT三维结构预测的。因此，对于亚科Iα天冬氨酸和芳香族氨基转移酶的酶活性必不可少的两个严格保守的精氨酸中的只有一个对克鲁维酵母的TAT活性至关重要。

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