NMR line shape analysis of a multi-state ligand binding mechanism in chitosanase
- 716 Downloads
Chitosan interaction with chitosanase was examined through analysis of spectral line shapes in the NMR HSQC titration experiments. We established that the substrate, chitosan hexamer, binds to the enzyme through the three-state induced-fit mechanism with fast formation of the encounter complex followed by slow isomerization of the bound-state into the final conformation. Mapping of the chemical shift perturbations in two sequential steps of the mechanism highlighted involvement of the substrate-binding subsites and the hinge region in the binding reaction. Equilibrium parameters of the three-state model agreed with the overall thermodynamic dissociation constant determined by ITC. This study presented the first kinetic evidence of the induced-fit mechanism in the glycoside hydrolases.
KeywordsLine shape analysis NMR Chitosanase Chitosan Induced fit Exchange regime HSQC titration IDAP TITAN
This work was supported by “Strategic Project to Support the Formation of Research Bases at Private Universities: Matching Fund Subsidy from MEXT (Ministry of Education, Culture, Sports, Science and Technology), 2011–2015 (S1101035) and partially supported by the Platform Project for Supporting in Drug Discovery and Life Science Research (Platform for Drug Discovery, Informatics, and Structural Life Science from Japan Agency for Medical Research and development (AMED) to TF. Work at Université de Sherbrooke was sustained by a Discovery Grant from the Natural Science and Engineering Research Council of Canada to RB. ELK acknowledges Committee on Research (COR) Summer Faculty Fellowship 2012 from Marquette University. SS was supported by a Research Fellowship for Young Scientists from Japan Society for the Promotion of Science (25-3639).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
- Akaike H (1973) Information theory and an extension of the maximum likelihood principle. In: Petrov BN, Csaki BF (eds) Second International Symposium on Information Theory. Academiai Kiado, Budapest, pp 267–281Google Scholar
- Dubeau MP, Ghinet MG, Jacques PE, Clermont N, Beaulieu C, Brzezinski R (2009) Cytosine deaminase as a negative selection marker for gene disruption and replacement in the genus Streptomyces and other actinobacteria. Appl Environ Microbiol 75:1211–1214. doi: 10.1128/AEM.02139-08 CrossRefGoogle Scholar
- Goddard TD, Kneller DG (1997) SPARKY 3; http://www.cgl.ucsf.edu/home/sparky/. University of California, San Francisco
- Kaplan JI, Fraenkel G (1980) NMR of chemically exchanging systems. Academic Press, CambridgeGoogle Scholar
- Monaghan RL, Eveleigh DE, Tewari RP, Reese ET (1973) Chitosanase, a novel enzyme. Nature 245:78–80Google Scholar
- Motulsky H, Christopoulos A (2004) Fitting models to biological data using linear and nonlinear regression: a practical guide to curve fitting. 1st edn. Oxford University Press, USA. http://www.graphpad.com/manuals/prism4/RegressionBook.pdf
- Page N, Kluepfel D, Shareck F, Morosoli R (1996) Effect of signal peptide alterations and replacement on export of xylanase A in Streptomyces lividans. Appl Environ Microbiol 62:109–114Google Scholar
- Taylor JR (1997) An introduction to error analysis. 2nd edn. University Science Books, CaliforniaGoogle Scholar
- Wang Y, Zhou P, Yu J, Pan X, Wang P, Lan W, Tao S (2007) Antimicrobial effect of chitooligosaccharides produced by chitosanase from Pseudomonas CUY8. Asia Pac J Clin Nutr 16(Suppl 1):174–177Google Scholar