Structural and Kinetic Characterizations of the Polysialic Acid O-Acetyltransferase OatWY from Neisseria meningitidis

The neuroinvasive pathogen Neisseria meningitidis has 13 capsular serogroups, but the majority of disease is caused by only 5 of these. Groups B, C, Y, and W-135 all display a polymeric sialic acid-containing capsule that provides a means for the bacteria to evade the immune response during infection by mimicking host sialic acid-containing cell surface structures. These capsules in serogroups C, Y, and W-135 can be further acetylated by a sialic acid-specific O-acetyltransferase, a modification that correlates with decreased immunoreactivity and increased virulence. In N. meningitidis serogroup Y, the O-acetylation reaction is catalyzed by the enzyme OatWY, which we show has clear specificity toward the serogroup Y capsule ([Glc-(α1→4)-Sia]_n). To understand the underlying molecular basis of this process, we have performed crystallographic analysis of OatWY with bound substrate as well as determined kinetic parameters of the wild type enzyme and active site mutants. The structure of OatWY reveals an intimate homotrimer of left-handed β-helix motifs that frame a deep active site cleft selective for the polysialic acid-bearing substrate. Within the active site, our structural, kinetic, and mutagenesis data support the role of two conserved residues in the catalytic mechanism (His-121 and Trp-145) and further highlight a significant movement of Tyr-171 that blocks the active site of the enzyme in its native form. Collectively, our results reveal the first structural features of a bacterial sialic acid O-acetyltransferase and provide significant new insight into its catalytic mechanism and specificity for the capsular polysaccharide of serogroup Y meningococci.The bacterial pathogen Neisseria meningitidis is a major cause of life-threatening neuroinvasive meningitis in humans (1). In the United States, 75% of bacterial meningitis infections are caused by serogroup C, Y, or W-135 (2). In particular, the proportion of meningococcal infection occurrences in the United States caused by the group Y meningococci has increased significantly from 2% during 1989–1991 to 37% during 1997–2002 (2). Vaccines based on the capsular polysaccharide have been developed for groups A/C/Y/W-135 (2), and the introduction of a group C conjugate vaccine has reduced the incidence and carriage of the C serogroup significantly (3). Although these vaccines are working, they do not yet provide complete protection from meningococcal disease (4).The capsular polysaccharides of N. meningitidis are classified into 13 distinct serogroups based on their chemical structures (5). The capsules of serogroup B and C are homopolymers composed of α-2,8- or α-2,9-linked sialic acid, respectively, whereas serogroup Y and W-135 are heteropolymers of an α-2,6-linked sialic acid on glucose (Y) or galactose (W-135) (6, 7). N. meningitidis group B polysialic acid shares a biochemical epitope with the polysialylated form of the neural cell adhesion molecule of humans (8, 9). Because of this molecular mimicry of the polysialic acid-neural cell adhesion molecule, the bacterial capsular polysaccharide is thus considered a major virulence factor of N. meningitidis (5, 10).Serogroup C, Y, and W-135 of N. meningitidis modify their sialic acid capsules by O-acetylation of the sialic acid (11). Sialic acid is acetylated at the C-7 or C-8 position hydroxyl group in serogroup C, whereas the C-7 or C-9 position is acetylated in serogroup W-135 and Y (11). The O-acetylation of sialic acids is known to alter the physicochemical properties of the polysaccharide capsule (12). In addition, there is growing evidence that O-acetylation of the polysaccharide enhances bacterial pathogenesis by masking the protective epitope in the polysaccharide (13–16). For these reasons, considerable effort has been expended to identify and characterize sialic acid O-acetyltransferases in pathogenic bacteria.Recently, the sialic acid-specific O-acetyltransferases from group B Streptococcus, Campylobacter jejuni, Escherichia coli K1, and N. meningitidis serogroup C have been identified (17–20) with the latter two variants being the only ones to be characterized biochemically (21–23). These studies showed that bacterial sialic acid-specific O-acetyltransferases utilize an acetyl-CoA cofactor as a donor for the acetylation of their capsular sialic acid acceptor substrates (Fig. 1) and identified essential amino acid residues for potential catalytic roles in activity (22, 23). Although the gene encoding the capsule-specific O-acetyltransferase in N. meningitidis serogroup Y (known as OatWY) has been identified, biochemical characterization of the enzyme has not yet been reported. Furthermore, the lack of structural information on a sialic acid O-acetyltransferase from any bacterial species has hampered our ability to further understand the mode of substrate binding, specificity, and catalytic mechanism of this important sialic acid-modifying family.Open in a separate windowFIGURE 1.Reaction scheme of the OatWY-catalyzed O-acetyltransferase. Although acetylation of both the O-7 and O-9 hydroxyl group of the N. meningitidis serogroup Y polysialic acid has been implied through NMR analysis of the corresponding bacterial capsule (11), for simplicity only the O-9 transfer is shown here.Here we report the first kinetic and structural analysis of polysialic acid O-acetyltransferase OatWY from N. meningitidis serogroup Y in complex with either CoA, acetyl-CoA, or S-(2-oxopropyl)-CoA, which is a nonhydrolyzable acetyl-CoA substrate analog. Collectively, this study significantly contributes to our understanding of bacterial polysialic acid O-acetyltransferases, providing valuable insight into how capsular polysaccharide is acetylated in pathogenic bacteria.