Abstract：
Many bacteria glycosylate flagellin on serine or threonine residues using pseudaminic acid (Pse) or other sialic acid-like donor sugars. Successful reconstitution of Pse-dependent sialylation by the conserved Maf-type flagellin glycosyltransferase (fGT) may require (a) missing component(s). Here, we characterize both Maf paralogs in the Gram-negative bacterium Shewanella oneidensis MR-1 and reconstitute Pse-dependent glycosylation in heterologous hosts. Remarkably, we uncovered distinct acceptor determinants and target specificities for each Maf. Whereas Maf-1 uses its C-terminal tetratricopeptide repeat (TPR) domain to confer flagellin acceptor and O-glycosylation specificity, Maf-2 requires the newly identified conserved specificity factor, glycosylation factor for Maf (GlfM), to form a ternary complex with flagellin. GlfM orthologs are co-encoded with Maf-2 in Gram-negative and Gram-positive bacteria and require an invariant aspartate in their four-helix bundle to function with Maf-2. Thus, convergent fGT evolution underlies distinct flagellin-binding modes in tripartite versus bipartite systems and, consequently, distinct O-glycosylation preferences of acceptor serine residues with Pse.
摘要：
许多细菌使用假氨基酸(Pse)或其他唾液酸样供体糖在丝氨酸或苏氨酸残基上糖基化鞭毛蛋白。通过保守的Maf型鞭毛蛋白糖基转移酶(fGT)成功重建Pse依赖性唾液酸化可能需要(a)缺失组分。这里,我们表征了革兰氏阴性细菌ShewanellaoneidensisMR-1中的两个Maf旁系同源物，并在异源宿主中重建了Pse依赖性糖基化。值得注意的是,我们发现了每个Maf的不同受体决定因素和靶标特异性。而Maf-1使用其C末端四肽重复（TPR）结构域赋予鞭毛蛋白受体和O-糖基化特异性，Maf-2需要新鉴定的保守特异性因子，糖基化因子Maf（GlfM），与鞭毛蛋白形成三元复合物。在革兰氏阴性和革兰氏阳性细菌中，GlfM直向同源物与Maf-2共同编码，并且在其四螺旋束中需要不变的天冬氨酸才能与Maf-2一起发挥作用。因此，融合的fGT进化是三方系统和双向系统中不同的鞭毛蛋白结合模式的基础，因此,受体丝氨酸残基与Pse的不同O-糖基化偏好。
