Di-N-acetylated uronic acid residues are unique sugar moieties observed in the lipopolysaccharides (LPS) of respiratory pathogens including several serotypes of Pseudomonas aeruginosa and several species of Bordetella. WbpD of P. aeruginosa PAO1 (serotype O5) is a putative 3-N-acetyltransferase that has been implicated in the biosynthesis of UDP-2,3-diacetamido-2,3-dideoxy-d-mannuronic acid [UDP-d-Man(2NAc3NAc)A], a precursor for the d-Man(2NAc3NAc)A residues in the B-band O antigen of this bacterium. A chromosomal knockout mutant of wbpD is incapable of producing either long-chain B-band O antigen (> or = 2 repeating units) or semi-rough LPS (lipid A-core + one repeat). Adding wbpD in trans restored LPS production to the wild-type level; this indicates that wbpD is important for biosynthesis of individual B-band O-antigen repeating units. WbpD contains left-handed beta-helical (LbetaH) structure as observed by Conserved Domain analysis and in silico secondary and tertiary structure predictions. This feature suggested that WbpD belongs to the hexapeptide acyltransferase (HexAT) superfamily of enzymes. WbpD was overexpressed as an N-terminally histidine-tagged fusion protein (His6-WbpD) and purified to > 95% purity. The protein was subjected to Far-UV circular dichroism spectroscopy, and the data revealed that WbpD contains left-handed helical structure, which substantiated in silico predictions made earlier. Results from SDS-PAGE, matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS), and gel filtration analyses indicated that His6-WbpD has trimeric organization, consistent with the quaternary structure of HexATs. The binding of acetyl-CoA by WbpD was demonstrated by MALDI-TOF MS, suggesting that WbpD is an acetyltransferase that utilizes a direct-transfer reaction mechanism. Incubation of WbpD with acetyl-CoA significantly enhanced the stability of the protein and prevented precipitation over a course of 14 days. As a substrate for studying the enzymatic activity of WbpD is unavailable at present, a structure-based model for the LbetaH domain of WbpD was generated. Comparisons between this model and the LbetaH domains of known HexATs suggested that Lys136 plays a role in acetyl-CoA binding. A K136A site-directed mutant construct could only partially complement the wbpD knockout, and this mutation also reduced the stabilizing effects of acetyl-CoA, while a K136R mutation showed no discernible effect on complementation of the wbpD mutant or the stabilizing effects of acetyl-CoA on the purified mutant protein. A modified pathway was proposed for the biosynthesis of UDP-d-Man(2NAc3NAc)A, in which WbpD is involved in the catalysis of the fourth step by acting as a UDP-2-acetamido-3-amino-2,3-dideoxy-d-glucuronic acid 3-N-acetyltransferase.