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首页> 外文期刊>Biochemistry >Contributions of Unique Active Site Residues of Eukaryotic UDP-Galactopyranose Mutases to Substrate Recognition and Active Site Dynamics
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Contributions of Unique Active Site Residues of Eukaryotic UDP-Galactopyranose Mutases to Substrate Recognition and Active Site Dynamics

机译：真核UDP-半乳糖苷酶的独特活性位点残基对底物识别和活性位点动力学的贡献

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UDP-galactopyranose mutase (UGM) catalyzes the interconversion between UDP-galactopyranose and UDP-galactofuranose. Absent in humans, galactofuranose is found in bacterial and fungal cell walls and is a cell surface virulence factor in protozoan parasites. For these reasons, UGMs are targets for drug discovery. Here, we report a mutagenesis and structural study of the UGMs from Aspergillus fumigatus and Trypanosoma cruzi focused on active site residues that are conserved in eukaryotic UGMs but are absent or different in bacterial UGMs. Kinetic analysis of the variants F66A, Y104A, Q107A, N207A, and Y317A (A. fumigatus numbering) show decreases in k(cat)/K-M values of 200-1000-fold for the mutase reaction. In contrast, none of the mutations significantly affect the kinetics of enzyme activation by NADPH. These results indicate that the targeted residues are important for promoting the transition state conformation for UDP-galactofuranose formation. Crystal structures of the A. fumigatus mutant enzymes were determined in the presence and absence of UDP to understand the structural consequences of the mutations. The structures suggest important roles for Asn207 in stabilizing the closed active site, and Tyr317 in positioning of the uridine ring. Phe66 and the corresponding residue in Mycobacterium tuberculosis UGM (His68) play a role as the backstop, stabilizing the galactopyranose group for nucleophilic attack. Together, these results provide insight into the essentiality of the targeted residues for realizing maximal catalytic activity and a proposal for how conformational changes that close the active site are temporally related and coupled together.
机译：UDP-吡喃半乳糖突变酶（UGM）催化UDP-吡喃半乳糖和UDP-呋喃半乳糖之间的相互转化。半乳糖呋喃糖在人类中不存在，被发现在细菌和真菌的细胞壁中，并且是原生动物寄生虫中的一种细胞表面毒力因子。由于这些原因，UGM是药物发现的目标。在这里，我们报道了来自烟曲霉和克氏锥虫的UGM的诱变和结构研究，其重点是在真核UGM中保守但在细菌UGM中不存在或不同的活性位点残基。变体F66A，Y104A，Q107A，N207A和Y317A（烟曲霉）的动力学分析显示，突变酶反应的k（cat）/ K-M值降低了200-1000倍。相反，没有一个突变显着影响NADPH激活酶的动力学。这些结果表明，靶向残基对于促进UDP-半乳糖呋喃糖形成的过渡态构象很重要。在存在和不存在UDP的情况下确定烟曲霉突变酶的晶体结构，以了解突变的结构后果。这些结构表明Asn207在稳定封闭的活性位点和Tyr317在尿苷环的定位中起重要作用。 Phe66和结核分枝杆菌UGM（His68）中的相应残基起着支撑作用，稳定了吡喃半乳糖基团，以进行亲核攻击。在一起，这些结果提供了对于实现最大催化活性的目标残基的必要性的见解，并提出了关于封闭活性位点的构象变化如何在时间上相关并耦合在一起的建议。

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