Conflict of Interests: The authors declare no conflict of interests.
Substrate-mediated control of the conformation of an ancillary domain delivers a competent catalytic site for N-acetylneuraminic acid synthase
Article first published online: 16 APR 2014
© 2014 Wiley Periodicals, Inc.
Proteins: Structure, Function, and Bioinformatics
Volume 82, Issue 9, pages 2054–2066, September 2014
How to Cite
Joseph, D. D. A., Jiao, W., Kessans, S. A. and Parker, E. J. (2014), Substrate-mediated control of the conformation of an ancillary domain delivers a competent catalytic site for N-acetylneuraminic acid synthase. Proteins, 82: 2054–2066. doi: 10.1002/prot.24558
- Issue published online: 21 AUG 2014
- Article first published online: 16 APR 2014
- Accepted manuscript online: 14 MAR 2014 03:23AM EST
- Manuscript Accepted: 4 MAR 2014
- Manuscript Revised: 14 FEB 2014
- Manuscript Received: 9 JAN 2014
- Maurice Wilkins Centre for Molecular Biodiscovery
- College of Science, University of Canterbury
- antifreeze protein-like domain;
- N-acetylneuraminic acid;
- sialic acid;
- Neisseria meningitidis;
- TIM barrel;
- quaternary structure
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. Proteins 2014; 82:2054–2066. © 2014 Wiley Periodicals, Inc.