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-beta-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 alpha,beta 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.
Morrison
James P
JP
Department of Chemistry, University of British Columbia, Vancouver, BC, Canada V6T 1Z1.
Schoenhofen
Ian C
IC
Tanner
Martin E
ME
D001224:Aspartic Acid / Q000737:chemistry; D016123:Campylobacter jejuni / Q000378:metabolism; D002384:Catalysis; D016480:Helicobacter pylori / Q000378:metabolism; D006836:Hydro-Lyases / Q000138:chemical synthesis / Q000737:chemistry*; D007659:Ketones / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010100:Oxygen / Q000737:chemistry; D012997:Solvents / Q000737:chemistry; D013400:Sugar Acids / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000737:chemistry*; D014867:Water / Q000737:chemistry
C513444:5,7-diacetamido-3,5,7,9-tetradeoxynonulosonic acid; D007659:Ketones; D012997:Solvents; D013400:Sugar Acids; D014867:Water; D001224:Aspartic Acid; D014443:Tyrosine; D014537:Uridine Diphosphate N-Acetylglucosamine; D006836:Hydro-Lyases; C533785:UDP-N-acetylglucosamine 5-inverting 4,6-dehydratase, Campylobacter jejuni; D008239:Lysine; D010100:Oxygen