Flügge Friedemann, Peters Thomas
Institute for Chemistry and Metabolomics, Center for Structural and Cell Biology in Medicine, University of Lübeck, Ratzeburger Allee 160, 23562, Lübeck, Germany.
J Biomol NMR. 2018 Apr;70(4):245-259. doi: 10.1007/s10858-018-0183-4. Epub 2018 Apr 26.
Human blood group A and B glycosyltransferases (GTA, GTB) are highly homologous glycosyltransferases. A number of high-resolution crystal structures is available showing that these enzymes convert from an open conformation into a catalytically active closed conformation upon substrate binding. However, the mechanism of glycosyltransfer is still under debate, and the precise nature as well as the time scales of conformational transitions are unknown. NMR offers a variety of experiments to shine more light on these unresolved questions. Therefore, in a first step we have assigned all methyl resonance signals in MILVA labeled samples of GTA and GTB, still a challenging task for 70 kDa homodimeric proteins. Assignments were obtained from methyl-methyl NOESY experiments, and from measurements of lanthanide-induced pseudocontact shifts (PCS) using high resolution crystal structures as templates. PCSs and chemical shift perturbations, induced by substrate analogue binding, suggest that the fully closed state is not adopted in the presence of lanthanide ions.
人类血型A和B糖基转移酶（GTA、GTB）是高度同源的糖基转移酶。已有多个高分辨率晶体结构表明，这些酶在底物结合时从开放构象转变为具有催化活性的封闭构象。然而，糖基转移的机制仍存在争议，构象转变的确切性质以及时间尺度尚不清楚。核磁共振（NMR）提供了多种实验来更深入地了解这些未解决的问题。因此，第一步我们在GTA和GTB的MILVA标记样品中归属了所有甲基共振信号，对于70 kDa的同二聚体蛋白来说，这仍然是一项具有挑战性的任务。通过甲基-甲基NOESY实验以及使用高分辨率晶体结构作为模板测量镧系元素诱导的伪接触位移（PCS）来获得归属。底物类似物结合诱导的PCS和化学位移扰动表明，在存在镧系离子的情况下，不会采用完全封闭状态。
