Schoenhofen Ian C, McNally David J, Brisson Jean-Robert, Logan Susan M
Institute for Biological Sciences, National Research Council, Ottawa, Ontario, Canada.
Glycobiology. 2006 Sep;16(9):8C-14C. doi: 10.1093/glycob/cwl010. Epub 2006 Jun 3.
Flagellin glycosylation is a necessary modification allowing flagellar assembly, bacterial motility, colonization, and hence virulence for the gastrointestinal pathogen Helicobacter pylori [Josenhans, C., Vossebein, L., Friedrich, S., and Suerbaum, S. (2002) FEMS Microbiol. Lett., 210, 165-172; Schirm, M., Schoenhofen, I.C., Logan, S.M., Waldron, K.C., and Thibault, P. (2005) Anal. Chem., 77, 7774-7782]. A causative agent of gastric and duodenal ulcers, H. pylori, heavily modifies its flagellin with the sialic acid-like sugar 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-alpha-l-manno-nonulosonic acid (pseudaminic acid). Because this sugar is unique to bacteria, its biosynthetic pathway offers potential as a novel therapeutic target. We have identified six H. pylori enzymes, which reconstitute the complete biosynthesis of pseudaminic acid, and its nucleotide-activated form CMP-pseudaminic acid, from UDP-N-acetylglucosamine (UDP-GlcNAc). The pathway intermediates and final product were identified from monitoring sequential reactions with nuclear magnetic resonance (NMR) spectroscopy, thereby confirming the function of each biosynthetic enzyme. Remarkably, the conversion of UDP-GlcNAc to CMP-pseudaminic acid was achieved in a single reaction combining six enzymes. This represents the first complete in vitro enzymatic synthesis of a sialic acid-like sugar and sets the groundwork for future small molecule inhibitor screening and design. Moreover, this study provides a strategy for efficient large-scale synthesis of novel medically relevant bacterial sugars that has not been attainable by chemical methods alone.
鞭毛蛋白糖基化是一种必要的修饰，它允许鞭毛组装、细菌运动、定殖，从而使胃肠道病原体幽门螺杆菌具有致病性[约森汉斯，C.，沃斯贝恩，L.，弗里德里希，S.，和苏尔鲍姆，S.（2002年）《FEMS微生物学快报》，210，165 - 172；施irm，M.，舍恩霍芬，I.C.，洛根，S.M.，沃尔德伦，K.C.，和蒂博，P.（2005年）《分析化学》，77，7774 - 7782]。作为胃溃疡和十二指肠溃疡的病原体，幽门螺杆菌用类似唾液酸的糖5,7 - 二乙酰氨基 - 3,5,7,9 - 四脱氧 - l - 甘油 - α - l - 甘露糖 - 壬酮糖醛酸（假氨基糖酸）对其鞭毛蛋白进行大量修饰。由于这种糖是细菌特有的，其生物合成途径具有作为新型治疗靶点的潜力。我们已经鉴定出六种幽门螺杆菌酶，它们从UDP - N - 乙酰葡糖胺（UDP - GlcNAc）重构了假氨基糖酸及其核苷酸活化形式CMP - 假氨基糖酸的完整生物合成。通过用核磁共振（NMR）光谱监测连续反应鉴定了途径中间体和最终产物，从而证实了每种生物合成酶的功能。值得注意的是，在一个结合六种酶的单一反应中实现了从UDP - GlcNAc到CMP - 假氨基糖酸的转化。这代表了首次在体外完全酶促合成类似唾液酸的糖，并为未来小分子抑制剂的筛选和设计奠定了基础。此外，这项研究提供了一种高效大规模合成新型医学相关细菌糖的策略，这是仅靠化学方法无法实现的。
