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- Title
Determination of thioxylo-oligosaccharide binding to family 11 xylanases using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry and X-ray crystallography.
- Authors
Jänis, Janne; Hakanpää, Johanna; Hakulinen, Nina; Ibatullin, Farid M.; Hoxha, Antuan; Derrick, Peter J.; Rouvinen, Juha; Vainiotalo, Pirjo
- Abstract
Noncovalent binding of thioxylo-oligosaccharide inhibitors, methyl 4-thio-α-xylobioside (S-Xyl2-Me), methyl 4,4II-dithio-α-xylotrioside (S-Xyl3-Me), methyl 4,4II,4III-trithio-α-xylotetroside (S-Xyl4-Me), and methyl 4,4II,4III,4IV-tetrathio-α-xylopentoside (S-Xyl5-Me), to three family 11 endo-1,4-β-xylanases fromTrichoderma reesei(TRX I and TRX II) andChaetomium thermophilum(CTX) was characterized using electrospray ionization Fourier transform ion cyclotron resonance (FT-ICR) MS and X-ray crystallography. Ultra-high mass-resolving power and mass accuracy inherent to FT-ICR allowed mass measurements for noncovalent complexes to within |ΔM|average of 2 p.p.m. The binding constants determined by MS titration experiments were in the range 104−103 M−1, decreasing in the series of S-Xyl5-Me ≥ S-Xyl4-Me > S-Xyl3-Me. In contrast, S-Xyl2-Me did not bind to any xylanase at the initial concentration of 5–200 µm, indicating increasing affinity with increasing number of xylopyranosyl units, with a minimum requirement of three. The crystal structures of CTX–inhibitor complexes gave interesting insights into the binding. Surprisingly, none of the inhibitors occupied any of the aglycone subsites of the active site. The binding to only the glycone subsites is nonproductive for catalysis, and yet this has also been observed for other family 11 xylanases in complex withβ-d-xylotetraose[Wakarchuk WW, Campbell RL, Sung WL, Davoodi J&Makoto Y (1994)Protein Sci3, 465–475, and Sabini E, Wilson KS, Danielsen S, Schülein M&Davies GJ (2001)Acta CrystallogrD57, 1344–1347]. Therefore, the role of the aglycone subsites remains controversial despite their obvious contribution to catalysis.
- Subjects
OLIGOSACCHARIDES; GLUCOSIDES; MONOSACCHARIDES; ELECTROSPRAY ionization mass spectrometry; CYCLOTRONS; SACCHARIDES; CRYSTALLOGRAPHY
- Publication
FEBS Journal, 2005, Vol 272, Issue 9, p2317
- ISSN
1742-464X
- Publication type
Article
- DOI
10.1111/j.1742-4658.2005.04659.x