Department of Plant and Microbial Biology, University of California, Berkeley, California, USA; Feedstocks Division, Joint BioEnergy Institute, Emeryville, California, USA; Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA; Center for Biomolecular Structure, Function and Application, Suranaree University of Technology, Nakhon Ratchasima, Thailand.
Biofuels and Bioproducts Division, Joint BioEnergy Institute, Emeryville, California, USA.
J Biol Chem. 2024 Sep;300(9):107602. doi: 10.1016/j.jbc.2024.107602. Epub 2024 Jul 24.
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.
糖基化是植物微调小分子代谢物性质以影响其生物活性、运输和储存的主要策略。在生物技术和医学中，许多糖苷也被用于人类健康，因此糖基化也很重要。小分子糖基化主要由家族 1 糖基转移酶完成。在这里，我们报告了 Pilosella officinarum 中的家族 1 GT 酶 UGT95A1 的结构和生化研究，该酶对黄酮类受体底物木樨草素的 3'-O 位置表现出强烈的、不寻常的区域特异性。我们获得了一个apo 晶体结构，以帮助驱动一系列结合位点突变体的分析，揭示尽管大多数残基对突变具有耐受性，但关键残基 M145 和 D464 对整体糖基化活性很重要。有趣的是，E347 对于维持对 3'-O 糖基化的强烈偏好至关重要，而 R462 可以突变以提高区域选择性。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，该酶包含大的、高度动态的无规卷曲区域。我们表明，虽然蛋白质的大多数无规卷曲区域对催化没有影响，但在研究的同源物中保守的无规卷曲区域对于酶的整体效率和区域选择性都很重要。本报告代表了对具有独特底物区域特异性的家族 1 GT 酶的全面深入分析，可为酶功能预测和工程提供基础。
