Biosynthesis of UDP-2-acetamido-4-formamido-2,4,6-trideoxy-hexose by WekG, WekE, WekF, and WekD: Enzymes in the Wek pathway responsible for O-antigen Assembly in Escherichia coli O119

Abstract

Considering the importance of bacterial glycoconjugates on virulence and host mimicry, there is a need to better understand the biosynthetic pathways of these unusual sugars to identify critical targets involved in bacterial pathogenesis. In this report, we describe the cloning, overexpression, purification, and biochemical characterization of the four central enzymes in the biosynthesis pathway for UDP-2-acetamido-4-formamido-2,4,6-trideoxy-hexose, WekG, WekE, WekF, and WekD. Product peaks from enzyme-substrate reactions were detected by using a combination of capillary electrophoresis (CE) and electrospray ionization-mass spectrometry (ESI-MS). Putative enzyme assignments were provided by protein sequence analysis. Combined with the mass spectrometric characterization of pathway intermediates, we propose a biosynthetic pathway for UDP-2-acetamido-4-formamido-2,4,6-trideoxy-hexose. This process involves C-4, C-6 dehydration, C-4 amination, and formylation. CID-ESI-MSn result confirmed that the final product is a 4 formamido derivative too rather than the 3 formamido derivatives as reported earlier.