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首页> 外文期刊>FEBS Open Bio >Biochemical and structural characterization of tyrosine aminotransferase suggests broad substrate specificity and a two‐state folding mechanism in Leishmania donovani
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Biochemical and structural characterization of tyrosine aminotransferase suggests broad substrate specificity and a two‐state folding mechanism in Leishmania donovani

机译：酪氨酸转氨酶的生化和结构表征表明在利什曼原虫中广泛的底物特异性和两种状态的折叠机制

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Tyrosine aminotransferase (TAT) is an aminotransferase with broad substrate specificity that catalyzes the transamination of aromatic amino acids in Leishmania?donovani and plays a crucial role in the survival and pathogenicity of the parasite. In this study, we have biochemically characterized tyrosine aminotransferase from Leishmania?donovani using in?vitro and in?silico techniques. Leishmania?donovani tyrosine aminotransferase (LdTAT) was cloned into the pET28a(+) vector and expressed in the BL21 strain of Escherichia?coli . The Ni‐NTA‐purified protein was then characterized biochemically, and its various kinetic parameters were investigated. The apparent K subm/sub value for the tyrosine–pyruvate pair was determined to be 3.5?±?0.9?m m , and V submax/sub was analyzed to be at 11.7?±?1.5?μ m ·min.μgsup?1/sup. LdTAT was found to exhibit maximum activity at 50?°C and at a pH of 8.0. Cofactor identification for LdTAT showed that pyridoxal‐5‐phosphate (PLP) binds with a K subm/sub value of 23.59?±?3.99?μ m and that the phosphate group is vital for the activity of the enzyme. Sequence analysis revealed that S151, Y256, K286, and P291 are conserved residues and form hydrogen bonds with PLP. Urea‐based denaturation studies revealed a biphasic folding mechanism involving N→X→D states. Molecular dynamic simulations of modeled LdTAT at various conditions were performed to understand enzyme behavior and interactions at the molecular level. The biochemical and structural divergence between host and parasite TAT suggests the LdTAT has evolved to utilize pyruvate rather than α‐ketoglutarate as co‐substrate. Furthermore, our data suggest that LdTAT may be a potential drug target due to its divergence in structure and substrate specificity from the host.
机译：酪氨酸氨基转移酶（TAT）是一种具有广泛底物特异性的氨基转移酶，可催化利什曼原虫中的芳香族氨基酸的氨基转移，并在该寄生虫的存活和致病性中起关键作用。在这项研究中，我们已经使用体外和计算机技术对来自利什曼原虫donovani的酪氨酸氨基转移酶进行了生化表征。利什曼原虫？donovani酪氨酸氨基转移酶（LdTAT）被克隆到pET28a（+）载体中，并在大肠杆菌BL21菌株中表达。然后对Ni-NTA纯化的蛋白质进行生化表征，并研究其各种动力学参数。酪氨酸-丙酮酸盐对的表观K m 值确定为3.5？±？0.9？mm，而V max 分析为11.7？±？1.5 ？μm·min.μg？1 。发现LdTAT在50℃和pH为8.0时表现出最大的活性。 LdTAT的辅因子鉴定表明，吡ido醛-5-磷酸（PLP）以23.59？±？3.99？μm的K m 值结合，并且磷酸基团对该酶的活性至关重要。序列分析表明S151，Y256，K286和P291是保守残基，并与PLP形成氢键。基于尿素的变性研究揭示了涉及N→X→D状态的双相折叠机制。进行了在各种条件下模拟的LdTAT的分子动力学模拟，以了解酶的行为和分子水平上的相互作用。宿主和寄生虫TAT之间的生化和结构差异表明，LdTAT已经进化为利用丙酮酸而不是α-酮戊二酸作为共底物。此外，我们的数据表明，由于LdTAT与宿主的结构和底物特异性不同，它可能成为潜在的药物靶标。

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