Structural and mechanistic analysis of sialic acid synthase NeuB from Neisseria meningitidis in complex with Mn2+, phosphoenolpyruvate, and N-acetylmannosaminitol.
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 18O-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 A 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 carbonyl to the addition of PEP.
Gunawan
Jason
J
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3.
Simard
Dave
D
Gilbert
Michel
M
Lovering
Andrew L
AL
Wakarchuk
Warren W
WW
Tanner
Martin E
ME
Strynadka
Natalie C J
NC
D000595:Amino Acid Sequence; D001665:Binding Sites; D003461:Crystallography; D019281:Dimerization; D006595:Hexosamines / Q000378:metabolism*; D008345:Manganese / Q000378:metabolism*; D008969:Molecular Sequence Data; D009345:Neisseria meningitidis / Q000201:enzymology*; D007652:Oxo-Acid-Lyases / Q000737:chemistry* / Q000378:metabolism*; D010728:Phosphoenolpyruvate / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship
D006595:Hexosamines; D008345:Manganese; D010728:Phosphoenolpyruvate; C018730:N-acetylneuraminate synthase; D007652:Oxo-Acid-Lyases; C002022:N-acetylmannosamine