PDF(Pubmed)
Abstract：
Glycosylation is a predominant strategy plants use to fine-tune the properties of small molecule metabolites to affect their bioactivity, transport, and storage. It is also important in biotechnology and medicine as many glycosides are utilized in human health. Small molecule glycosylation is largely carried out by family 1 glycosyltransferases. Here, we report a structural and biochemical investigation of UGT95A1, a family 1 GT enzyme from Pilosella officinarum that exhibits a strong, unusual regiospecificity for the 3\'-O position of flavonoid acceptor substrate luteolin. We obtained an apo crystal structure to help drive the analyses of a series of binding site mutants, revealing that while most residues are tolerant to mutations, key residues M145 and D464 are important for overall glycosylation activity. Interestingly, E347 is crucial for maintaining the strong preference for 3\'-O glycosylation, while R462 can be mutated to increase regioselectivity. The structural determinants of regioselectivity were further confirmed in homologous enzymes. Our study also suggests that the enzyme contains large, highly dynamic, disordered regions. We showed that while most disordered regions of the protein have little to no implication in catalysis, the disordered regions conserved among investigated homologs are important to both the overall efficiency and regiospecificity of the enzyme. This report represents a comprehensive in-depth analysis of a family 1 GT enzyme with a unique substrate regiospecificity and may provide a basis for enzyme functional prediction and engineering.
摘要：
糖基化是植物用来微调小分子代谢物的特性以影响其生物活性的主要策略。运输,和存储。它在生物技术和医学中也很重要，因为许多糖苷被用于人类健康。小分子糖基化主要由家族1糖基转移酶进行。这里,我们报告了UGT95A1的结构和生化研究，UGT95A1是一种来自药霉的家族1GT酶，类黄酮受体底物木犀草素的3'-O位置异常的区域特异性。我们获得了apo晶体结构，以帮助驱动一系列结合位点突变体的分析，揭示了虽然大多数残基对突变具有耐受性，关键残基M145和D464对于总体糖基化活性是重要的。有趣的是,E347对于保持3'-O糖基化的强烈偏好至关重要，而R462可以突变以增加区域选择性。在同源酶中进一步证实了区域选择性的结构决定因素。我们的研究还表明，这种酶含有大量的，高度动态,无序的地区。我们表明，虽然蛋白质的大多数无序区域对催化几乎没有影响，所研究的同源物之间保守的无序区域对酶的整体效率和区域特异性都很重要。该报告代表了对具有独特底物区域特异性的家族1GT酶的全面深入分析，并可能为酶功能预测和工程提供基础。
