title: Substrate-mediated control of the conformation of an ancillary domain delivers a competent catalytic site for N-acetylneuraminic acid synthase.
abstract: N-Acetylneuraminic acid (NANA) is the most common naturally occurring sialic acid and plays a key role in the pathogenesis of a select number of neuroinvasive bacteria such as Neisseria meningitidis. NANA is synthesized in prokaryotes via a condensation reaction between phosphoenolpyruvate and N-acetylmannosamine. This reaction is catalyzed by a domain swapped, homodimeric enzyme, N-acetylneuraminic acid synthase (NANAS). NANAS comprises two distinct domains; an N-terminal catalytic (β/α)8 barrel linked to a C-terminal antifreeze protein-like (AFPL) domain. We have investigated the role of the AFPL domain by characterizing a truncated variant of NmeNANAS, which was discovered to be soluble yet inactive. Analytical ultracentrifugation and analytical size exclusion were used to probe the quaternary state of the NmeNANAS truncation, and revealed that loss of the AFPL domain destabilizes the dimeric form of the enzyme. The results from this study thereby demonstrate that the AFPL domain plays a critical role for both the catalytic function and quaternary structure stability of NANAS. Small angle X-ray scattering, molecular dynamics simulations, and amino acid substitutions expose a complex hydrogen-bonding relay, which links the roles of the catalytic and AFPL domains across subunit boundaries.
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: Sarah A
authors.initials: SA
authors.lastname: Kessans
authors.forename: Emily J
authors.initials: EJ
authors.lastname: Parker
mesh_terms: D001665:Binding Sites; D020134:Catalytic Domain; D002850:Chromatography, Gel; D018360:Crystallography, X-Ray; D006595:Hexosamines / Q000737:chemistry; D006860:Hydrogen Bonding; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D019158:N-Acetylneuraminic Acid / Q000096:biosynthesis; D009345:Neisseria meningitidis / Q000472:pathogenicity*; D007652:Oxo-Acid-Lyases / Q000378:metabolism*; D010728:Phosphoenolpyruvate / Q000737:chemistry; D020836:Protein Structure, Quaternary
chemical_list: D006595:Hexosamines; D010728:Phosphoenolpyruvate; C018730:N-acetylneuraminate synthase; D007652:Oxo-Acid-Lyases; D019158:N-Acetylneuraminic Acid; C002022:N-acetylmannosamine
keywords: N-acetylneuraminic acid; Neisseria meningitidis; TIM barrel; antifreeze protein-like domain; quaternary structure; sialic acid