Abstract
O-Acetylation of the capsular polysaccharide (CPS) of Neisseria meningitidis serogroup A (NmA) is critical for the induction of functional immune responses, making this modification mandatory for CPS-based anti-NmA vaccines. Using comprehensive NMR studies, we demonstrate that O-acetylation stabilizes the labile anomeric phosphodiester-linkages of the NmA-CPS and occurs in position C3 and C4 of the N-acetylmannosamine units due to enzymatic transfer and non-enzymatic ester migration, respectively. To shed light on the enzymatic transfer mechanism, we solved the crystal structure of the capsule O-acetyltransferase CsaC in its apo and acceptor-bound form and of the CsaC-H228A mutant as trapped acetyl-enzyme adduct in complex with CoA. Together with the results of a comprehensive mutagenesis study, the reported structures explain the strict regioselectivity of CsaC and provide insight into the catalytic mechanism, which relies on an unexpected Gln-extension of a classical Ser-His-Asp triad, embedded in an α/β-hydrolase fold.
Highlights
O-Acetylation of the capsular polysaccharide (CPS) of Neisseria meningitidis serogroup A (NmA) is critical for the induction of functional immune responses, making this modification mandatory for CPS-based anti-NmA vaccines
The NmA-CPS is joint by phosphodiester bridges[6,7] that are highly susceptible to hydrolysis at elevated temperatures, which has major implications for the shelf-life and storage conditions of anti-NmA vaccines in tropical and subtropical areas[4,8]
Supported by a comprehensive two-dimensional (2D) NMR characterization of the CPS, we demonstrate that the CsaC-mediated reaction is regioselective for O3 and that modification of O4 results from spontaneous O-acetyl migration
Summary
Spin systems present in both samples 7. Spin system 2 shows correlations similar to those observed for 1 (3OAc-ManNAc), including the characteristic H3 chemical shift of 5.18 ppm, indicating O-acetylation at C3 (Fig. 2d and Supplementary Fig. 4). Spin systems 5 and 9 were present in both samples (Fig. 2c, d) and were assigned to the β- and α-anomer of non-O-acetylated ManNAc at the reducing end, respectively, based on the similarity to published chemical shifts of free ManNAc20 (Supplementary Table 1). Spin system 6 originates from a terminal ManNAc with a phosphomonoester at C6 and represents the non-reducing end of the polymer as shown by a distinct 31P chemical shift of 0.63 ppm correlating to H6a/H6b of 6 in a 1H-31P HMBC (heteronuclear multiple bond correlation) spectrum (Fig. 2f and Supplementary Fig. 7). Anchoring of the guanidinium group of R148 by two flanking H-bonds (with Y144 and the phosphodiester between ring C and D) directs the central R148-O4 interaction away from the catalytic center, ensuring regioselective attack of O3
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