Mechanistic studies on PseB of pseudaminic acid biosynthesis: A UDP-N-acetylglucosamine 5-inverting 4,6-dehydratase

UDP-N-acetylglucosamine 5-inverting 4,6-dehydratase (PseB) is a unique sugar nucleotide dehydratase that inverts the C-5″ stereocentre during conversion of UDP-N-acetylglucosamine to UDP-2-acetamido-2,6-dideoxy-β-l-arabino-hexos-4-ulose. PseB catalyzes the first step in the biosynthesis of pseudaminic acid, which is found as a post-translational modification on the flagellin of Campylobacter jejuni and Helicobacter pylori. PseB is proposed to use its tightly bound NADP+ to oxidize UDP-GlcNAc at C-4″, enabling dehydration. The α,β unsaturated ketone intermediate is then reduced by delivery of the hydride to C-6″ and a proton to C-5″. Consistent with this, PseB from C. jejuni has been found to incorporate deuterium into the C-5″ position of product during catalysis in D2O. Likewise, PseB catalyzes solvent isotope exchange into the H-5″ position of product, and eliminates HF from the alternate substrate, UDP-6-deoxy-6-fluoro-GlcNAc. Mutants of the putative catalytic residues aspartate 126, lysine 127 and tyrosine 135 have severely compromised dehydratase, solvent isotope exchange, and HF elimination activities.