1XUZ

Crystal structure analysis of sialic acid synthase (NeuB)from Neisseria meningitidis, bound to Mn2+, Phosphoenolpyruvate, and N-acetyl mannosaminitol

Summary for 1XUZ

• Entry DOI: 10.2210/pdb1xuz/pdb
• Related: 1XUU
• Descriptor: polysialic acid capsule biosynthesis protein SiaC, 5-DEOXY-5-{[(1S)-1-HYDROXYETHYL]AMINO}-D-GLUCITOL, MANGANESE (II) ION, ... (5 entities in total)
• Functional Keywords: tim barrel, antifreeze-like domain, domain-swapped dimer, biosynthetic protein
• Biological source: Neisseria meningitidis
• Total number of polymer chains: 1
• Total formula weight: 38843.95

Authors

Gunawan, J.,Simard, D.,Gilbert, M.,Lovering, A.L.,Wakarchuk, W.W.,Tanner, M.E.,Strynadka, N.C. (deposition date: 2004-10-26, release date: 2004-11-02, Last modification date: 2024-02-14)

Primary citation

Gunawan, J.,Simard, D.,Gilbert, M.,Lovering, A.L.,Wakarchuk, W.W.,Tanner, M.E.,Strynadka, N.C.
Structural and mechanistic analysis of sialic acid synthase NeuB from Neisseria meningitidis in complex with Mn2+, phosphoenolpyruvate, and N-acetylmannosaminitol.
J.Biol.Chem., 280:3555-3563, 2005

PubMed 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 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.

PubMed: 15516336
DOI: 10.1074/jbc.M411942200

Experimental method

X-RAY DIFFRACTION (2.2 Å)