Bacterial β-Kdo glycosyltransferases represent a new glycosyltransferase family (GT99)

Abstract

Kdo (3-deoxy-D-manno-oct-2-ulosonic acid) is an eight-carbon sugar mostly confined to Gram-negative bacteria. It is often involved in attaching surface polysaccharides to their lipid anchors. α-Kdo provides a bridge between lipid A and the core oligosaccharide in all bacterial LPSs, whereas an oligosaccharide of β-Kdo residues links "group 2" capsular polysaccharides to (lyso)phosphatidylglycerol. β-Kdo is also found in a small number of other bacterial polysaccharides. The structure and function of the prototypical cytidine monophosphate-Kdo-dependent α-Kdo glycosyltransferase from LPS assembly is well characterized. In contrast, the β-Kdo counterparts were not identified as glycosyltransferase enzymes by bioinformatics tools and were not represented among the 98 currently recognized glycosyltransferase families in the Carbohydrate-Active Enzymes database. We report the crystallographic structure and function of a prototype β-Kdo GT from WbbB, a modular protein participating in LPS O-antigen synthesis in Raoultella terrigena. The β-Kdo GT has dual Rossmann-fold motifs typical of GT-B enzymes, but extensive deletions, insertions, and rearrangements result in a unique architecture that makes it a prototype for a new GT family (GT99). The cytidine monophosphate-binding site in the C-terminal α/β domain closely resembles the corresponding site in bacterial sialyltransferases, suggesting an evolutionary connection that is not immediately evident from the overall fold or sequence similarities. microbial glycobiology | 3-deoxy-D-manno-oct-2-ulosonic acid | Kdo | glycosyltransferase | polysaccharide

Biochemical Characterization of Bifunctional 3-Deoxy-β-d-manno-oct-2-ulosonic Acid (β-Kdo) Transferase KpsC from Escherichia coli Involved in Capsule Biosynthesis

“…The spectrum also contained a peak at m/z 718.30, which could be explained by traces of contaminating unreacted substrate 3, slow degradation of 4 during sample handling, and/or cleavage of (237) linkage because of in-source fragmentation of parent ion. Similar in-source fragmentation resulted in loss of terminal Kdo residue was reported earlier for a Kdo-(233)-Rha-(133)-GlcNAc trisaccharide derivative (5). TLC analysis of product 4 left over after ESI MS analysis confirmed that the amount of contaminating 3 (if present) is very low (Fig.…”

“…Regions corresponding to amino acids 2-675, 2-352, 303-675, and 353-675 of the protein were cloned into the pET28a(ϩ) vector generating proteins with C-terminal His 6 tags. The prototype WbbB ␤-Kdo GT99 enzyme contained conserved HP and QXXXD motifs that are essential for its catalytic activity (5 each domain of KpsC(2-675) were replaced with two alanine residues, and an additional construct was made with both domains mutated in this way. Independently, the predicted catalytic nucleophilic Asp was changed to Ala in both domains, separately and in tandem.…”

“…Recently, we reported the crystal structure of a prototype ␤-Kdo GT domain from a multidomain protein WbbB from Raoultella terrigena (5). The structure possesses dual Rossmann-fold motifs typically found in GTs with the widespread GT-B fold (6), including the ␣-Kdo transferase, WaaA (7).…”

“…ABC transporter-dependent pathways involving synthesis of a ␤-Kdo linker are found in prominent human pathogens, including E. coli, N. meningitidis, and Haemophilus influenzae (1,5). In E. coli, they are found in extra-intestinal isolates, including the globally disseminated multidrug-resistant ST131 group responsible for the majority of extra-intestinal infections by this species (9).…”

“…Moraxella nonliquefaciens and Kingella kingae), zoonotic pathogens (Pasteurella multocida) and pathogens of livestock (e.g. Actinobacillus pleuropneumoniae, Mannheimia hemolytica and P. multocida) (1,5,10). The distribution in human pathogens, and the importance of the capsule layers in the virulence of those bacteria, makes the KpsS and KpsC ␤-Kdo GTs attractive targets for small molecule inhibitors in anti-virulence strategies, where unmasked bacteria would be susceptible to host defenses.…”