Gram-negative bacterial viability is greatly reduced by the disruption of heptose sugar addition during the biosynthesis of lipopolysaccharide (LPS), an important bacterial outer membrane component. Heptosyltransferase I (HepI), a member of the GT-B structural subclass of glycosyltransferases, is therefore an essential enzyme for the biosynthesis of the LPS. The disruption of HepI also increases the susceptibility of bacteria to hydrophobic antibiotics, making HepI a potential target for drug development. In this work, the structural and dynamic properties of the catalytic cycle of HepI are explored. Previously, substrate-induced stabilization of HepI was observed and hypothesized to be assisted by interactions between the substrate and residues located on dynamic loops. Herein, positively charged amino acids were probed to identify binding partners of the negatively charged phosphates and carboxylates of Kdo₂-lipid A and its analogues. Mutant enzymes were characterized to explore changes in enzymatic activities and protein stability. Molecular modeling of HepI in the presence and absence of ligands was then performed with the wild type and mutant enzyme to allow determination of the relative change in substrate binding affinity resulting from each mutation. Together, these studies suggest that multiple residues are involved in mediating substrate binding, and a lack of additivity of these effects illustrates the functional redundancy of these binding interactions. The redundancy of residues mediating conformational transitions in HepI illustrates the evolutionary importance of these structural rearrangements for catalysis. This work enhances the understanding of HepI’s protein dynamics and mechanism and is a model for improving our understanding of glycosyltransferase enzymes.

中文翻译：

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相反吸引：底物与带正电荷的残基之间的相互作用引起的大肠杆菌庚糖基转移酶I构象变化。

脂多糖（LPS）（一种重要的细菌外膜成分）的生物合成过程中，添加庚糖的干扰会大大降低革兰氏阴性细菌的生存能力。因此，糖基转移酶的GT-B结构亚类成员庚基转移酶I（HepI）是LPS生物合成的必需酶。HepI的破坏还增加了细菌对疏水性抗生素的敏感性，使HepI成为药物开发的潜在目标。在这项工作中，探讨了HepI催化循环的结构和动力学性质。以前，观察到并假设底物诱导的HepI稳定，这是由于底物与位于动态环上的残基之间的相互作用所辅助。在这里₂-脂质A及其类似物。突变酶的特征在于探索酶活性和蛋白质稳定性的变化。然后用野生型和突变酶对在存在和不存在配体的情况下的HepI进行分子建模，从而确定每种突变导致的底物结合亲和力的相对变化。总之，这些研究表明，多个残基参与了底物结合的介导，而这些效应的加和性不足说明了这些结合相互作用的功能冗余。介导HepI中构象转变的残基的冗余性说明了这些结构重排对于催化的进化重要性。