Glycans, which are assembled through a process known as glycosylation, are critical for creating biological structures important for the function and vitality of bacterial cells. In order to study the synthesis of glycans, enzymes and intermediates involved in the various pathways are studied using analytical tools such as fluorescent isoprenoid probes as well as liquid chromatography-mass spectrometry (LC-MS) analysis. The research here focuses on an in vitro analysis of WecG, WecB, and WecC involved in the production of Enterobacterial common antigen (ECA) as well as glycolipid intermediates in the H. pylori biosynthetic pathway. UDP-ManNAcA, a substrate used by WecG, is not commercially available, so enzymatic synthesis has been attempted in the Troutman Lab utilizing two other ECA enzymes, WecB (an epimerase) and WecC (a dehydrogenase) with confirmation of activity through detection of UDP-N-acetylhexosaminuronic acid (HexNAcA) sugars by the development of a novel LC-MS method utilizing the properties of a Hydrophilic Interaction Liquid Chromatography-Zwitterionic (HILIC-Z) column for separation and detection of highly polar compounds. Through this method, it has been observed that WecC can produce UDP-GlcNAcA as well, a novel finding that has also resulted in difficulties in obtaining UDP-ManNAcA. This unexpected finding has led to probing WecC for specificity as well as a kinetic analysis. WecG’s activity as a N-acetyl-D-mannosaminuronic acid (ManNAcA) transferase has been successfully verified in vitro and is being probed for specificity. Proteins have begun to be expressed for studying the H. pylori glycosylation pathway and this research is currently in its nascent stages.
