Institut de Chimie Organique et Analytique (ICOA), UMR 7311 CNRS-Université d'Orléans, Université d'Orléans, Orléans Cedex 2, France; School of Pharmacy, Queen's University Belfast, Medical Biology Centre, Belfast, United Kingdom.
Institut de Chimie Organique et Analytique (ICOA), UMR 7311 CNRS-Université d'Orléans, Université d'Orléans, Orléans Cedex 2, France.
J Biol Chem. 2024 Mar;300(3):105747. doi: 10.1016/j.jbc.2024.105747. Epub 2024 Feb 13.
Glycosyltransferases (GT) catalyze the glycosylation of bioactive natural products, including peptides and proteins, flavonoids, and sterols, and have been extensively used as biocatalysts to generate glycosides. However, the often narrow substrate specificity of wild-type GTs requires engineering strategies to expand it. The GT-B structural family is constituted by GTs that share a highly conserved tertiary structure in which the sugar donor and acceptor substrates bind in dedicated domains. Here, we have used this selective binding feature to design an engineering process to generate chimeric glycosyltransferases that combine auto-assembled domains from two different GT-B enzymes. Our approach enabled the generation of a stable dimer with broader substrate promiscuity than the parent enzymes that were related to relaxed interactions between domains in the dimeric GT-B. Our findings provide a basis for the development of a novel class of heterodimeric GTs with improved substrate promiscuity for applications in biotechnology and natural product synthesis.
糖基转移酶（GT）催化生物活性天然产物（包括肽和蛋白质、类黄酮和固醇）的糖基化，已被广泛用作生物催化剂来生成糖苷。然而，野生型 GT 的底物特异性往往很窄，需要工程策略来扩展它。GT-B 结构家族由 GT 组成，它们具有高度保守的三级结构，其中糖供体和受体底物结合在专用结构域中。在这里，我们利用这种选择性结合的特点，设计了一种工程化的过程，来产生嵌合糖基转移酶，将来自两种不同 GT-B 酶的自动组装结构域组合在一起。我们的方法生成了一种稳定的二聚体，其底物的混杂性比相关的亲本酶更广，这与二聚体 GT-B 中结构域之间的松弛相互作用有关。我们的发现为开发一类新型的具有改进的底物混杂性的异源二聚体 GT 提供了基础，可用于生物技术和天然产物合成应用。
