title: Arg314 is essential for catalysis by N-acetyl neuraminic acid synthase from Neisseria meningitidis.
abstract: The sialic acid N-acetylneuraminic acid (NANA) has a key role in the pathogenesis of a select number of neuroinvasive bacteria such as Neisseria meningitidis. These pathogens coat themselves with polysialic acids, mimicking the exterior surface of mammalian cells and consequentially concealing the bacteria from the host's immune system. NANA is synthesized in bacteria by the homodimeric enzyme NANA synthase (NANAS), which catalyzes a condensation reaction between phosphoenolpyruvate (PEP) and N-acetylmannosamine (ManNAc). NANAS is closely related to the α-keto acid synthases 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase and 3-deoxy-d-manno-octulosonate 8-phosphate synthase. NANAS differs from these enzymes in that it contains an antifreeze protein like (AFPL) domain, which extends from the C-terminal of the (β/α)8 barrel containing the active site and contributes a highly conserved arginine (Arg314) into the active site of the opposing monomer chain. We have investigated the role of Arg314 in NmeNANAS through mutagenesis and a combination of kinetic and structural analyses. Using isothermal titration calorimetry and molecular modeling, we have shown that Arg314 is required for the catalytic function of NANAS and that the delocalized positively charged guanidinium functionality of this residue provides steering of the sugar substrate ManNAc for suitable placement in the active site and thus reaction with PEP.
authors.affiliation: Biomolecular Interaction Centre and Department of Chemistry, University of Canterbury , Christchurch, New Zealand.
authors.forename: Dmitri D A
authors.initials: DD
authors.lastname: Joseph
authors.forename: Wanting
authors.initials: W
authors.lastname: Jiao
authors.forename: Emily J
authors.initials: EJ
authors.lastname: Parker
mesh_terms: D001120:Arginine / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002151:Calorimetry / Q000379:methods; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D038541:Neisseria meningitidis, Serogroup B / Q000201:enzymology*; D007652:Oxo-Acid-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010728:Phosphoenolpyruvate / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation
chemical_list: D001426:Bacterial Proteins; D010728:Phosphoenolpyruvate; D001120:Arginine; C018730:N-acetylneuraminate synthase; D007652:Oxo-Acid-Lyases