The structure of UDP-N-acetylglucosamine 2-epimerase reveals homology to phosphoglycosyl transferases
The structure of UDP-N-acetylglucosamine 2-epimerase reveals homology to phosphoglycosyl transferases
复制标题
DOI：
10.1021/bi001627x
复制
发表时间：
2000-12-12
期刊：
影响因子：
2.9
通讯作者：
Strynadka, NCJ
中科院分区：
文献类型：
--
作者：
Campbell, RE;Mosimann, SC;Strynadka, NCJ
细菌UDP-N-乙酰氨基葡萄糖2-差向异构酶催化UDP-N-乙酰氨基葡萄糖(UDP-GlcNAc)在C-2可逆异构化，从而为细菌提供甘露糖残基的活性供体UDP-N-乙酰甘露糖胺(UDP-ManNAc)。ManNAc对细菌中的几个过程至关重要，包括形成19F和19A型肺炎链球菌等病原体的抗吞噬被膜多糖。我们测定了与UDP结合的UDP-GlcNAc 2-差向异构酶的X射线结构(2.5埃)，并鉴定出与糖原磷酸化酶和T4噬菌体β-葡萄糖基转移酶的结构同源性。与这些磷酸糖基转移酶的关系非常耐人寻味，因为它们的催化机制可能有相似之处。具体地说，这一观察结果与UDP-GlcNAc 2-差向异构酶催化的UDP与糖类中间体的消除和再加成可能通过具有显著氧碳正离子性质的过渡态进行的说法一致。同源二聚异构体由两个相似的α/β/α夹心结构域组成，活性部位位于结构域界面的深裂缝中。对不对称单位中的多个拷贝的比较表明，同向异构体可以经历10度的域间旋转，这与调控机制有关。基于结构的序列比对确定了活性中心中的几个碱性残基，它们可能参与了C-2上的质子转移或所提议的氧卡宾离子样过渡态的稳定。这一对细菌差向异构酶结构的认识适用于双功能哺乳动物UDP-GlcNAc“水解”2-差向异构酶/ManNAc激酶的同源N-末端结构域，该结构域催化唾液酸生物合成途径中的速率决定步骤。
Bacterial UDP-N-acetylglucosamine 2-epimerase catalyzes the reversible epimerization at C-2 of UDP-N-acetylglucosamine (UDP-GlcNAc) and thereby provides bacteria with UDP-N-acetylmannosamine (UDP-ManNAc), the activated donor of ManNAc residues. ManNAc is critical for several processes in bacteria, including formation of the antiphagocytic capsular polysaccharide of pathogens such as Streptococcus pneumoniae types 19F and 19A. We have determined the X-ray structure (2.5 Angstrom) of UDP-GlcNAc 2-epimerase with bound UDP and identified a previously unsuspected structural homology with the enzymes glycogen phosphorylase and T4 phage beta -glucosyltransferase. The relationship to these phosphoglycosyl transferases is very intriguing in terms of possible similarities in the catalytic mechanisms. Specifically, this observation is consistent with the proposal that the UDP-GlcNAc 2-epimerase-catalyzed elimination and re-addition of UDP to the glycal intermediate may proceed through a transition state with significant oxocarbenium ion-like character. The homodimeric epimerase is composed of two similar alpha/beta/alpha sandwich domains with the active site-located in the deep cleft at the domain interface. Comparison of the multiple copies in the asymmetric unit has revealed that the epimerase can undergo a 10 degrees interdomain rotation that is implicated in the regulatory mechanism. A structure-based sequence alignment has identified several basic residues in the active site that may be involved in the proton transfer at C-2 or stabilization of the proposed oxocarbenium ion-like transition state. This insight into the structure of the bacterial epimerase is applicable to the homologous N-terminal domain of the bifunctional mammalian UDP-GlcNAc "hydrolyzing" 2-epimerase/ManNAc kinase that catalyzes the rate-determining step in the sialic acid biosynthetic pathway.