Title | Structural and mechanistic analysis of sialic acid synthase NeuB from Neisseria meningitidis in complex with Mn2+ phosphoenolpyruvate, and N-acetylmannosaminitol |
Publication Type | Journal Article |
Year of Publication | 2005 |
Authors | Gunawan, J, Simard, D, Gilbert, M, Lovering, AL, Wakarchuk, WW, Tanner, ME, Strynadka, NCJ |
Journal | Journal of Biological Chemistry |
Volume | 280 |
Pagination | 3555-3563 |
Date Published | Feb |
Type of Article | Article |
ISBN Number | 0021-9258 |
Keywords | 3-DEOXY-D-ARABINO-HEPTULOSONATE-7-PHOSPHATE SYNTHASE, 3-DEOXY-D-MANNO-OCTULOSONATE-8-PHOSPHATE SYNTHASE, ACETYLNEURAMINIC ACID, BISUBSTRATE, CAMPYLOBACTER-JEJUNI, CATALYTIC MECHANISM, CELL GLYCOSYLATION, ENZYMATIC SYNTHESIS, ESCHERICHIA-COLI, INHIBITOR, KDO8P SYNTHASE |
Abstract | In Neisseria meningitidis and related bacterial pathogens, sialic acids play critical roles in mammalian cell immunity evasion and are synthesized by a conserved enzymatic pathway that includes sialic acid synthase (NeuB, SiaC, or SynC). NeuB catalyzes the condensation of phosphoenolpyruvate (PEP) and N-acetylmannosamine, directly forming N-acetylneuraminic acid (or sialic acid). In this paper we report the development of a coupled assay to monitor NeuB reaction kinetics and an O-18-labeling study that demonstrates the synthase operates via a C-O bond cleavage mechanism. We also report the first structure of a sialic acid synthase, that of NeuB, revealing a unique domain-swapped homodimer architecture consisting of a (beta/alpha)(8) barrel (TIM barrel)type fold at the N-terminal end and a domain with high sequence identity and structural similarity to the ice binding type III antifreeze proteins at the C-terminal end of the enzyme. We have determined the structures of NeuB in the malate-bound form and with bound PEP and the substrate analog N-acetylmannosaminitol to 1.9 and 2.2 Angstrom resolution, respectively. Typical of other TIM barrel proteins, the active site of NeuB is located in a cavity at the C-terminal end of the barrel; however, the positioning of the swapped antifreeze-like domain from the adjacent monomer provides key residues for hydrogen bonding with substrates in the active site of NeuB, a structural feature that leads to distinct modes of substrate binding from other PEP-utilizing enzymes that lack an analogous antifreeze-like domain. Our observation of a direct interaction between a highly ordered manganese and the N-acetylmannosaminitol in the NeuB active site also suggests an essential role for the ion as an electrophilic catalyst that activates the N-acetylmannosamine carbortyl to the addition of PEP. |
URL | <Go to ISI>://000226983900053 |