Premium
Determination of thioxylo‐oligosaccharide binding to family 11 xylanases using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry and X‐ray crystallography
Author(s) -
Jänis Janne,
Hakanpää Johanna,
Hakulinen Nina,
Ibatullin Farid M.,
Hoxha Antuan,
Derrick Peter J.,
Rouvinen Juha,
Vainiotalo Pirjo
Publication year - 2005
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2005.04659.x
Subject(s) - chemistry , fourier transform ion cyclotron resonance , electrospray ionization , mass spectrometry , crystallography , oligosaccharide , stereochemistry , aglycone , analytical chemistry (journal) , chromatography , glycoside , organic chemistry
Noncovalent binding of thioxylo‐oligosaccharide inhibitors, methyl 4‐thio‐α‐xylobioside (S‐Xyl2‐Me), methyl 4,4 II ‐dithio‐α‐xylotrioside (S‐Xyl3‐Me), methyl 4,4 II ,4 III ‐trithio‐α‐xylotetroside (S‐Xyl4‐Me), and methyl 4,4 II ,4 III ,4 IV ‐tetrathio‐α‐xylopentoside (S‐Xyl5‐Me), to three family 11 endo‐1,4‐β‐xylanases from Trichoderma reesei (TRX I and TRX II) and Chaetomium 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 10 4 −10 3 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 µ m , 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 with β‐ d ‐xylotetraose [Wakarchuk WW, Campbell RL, Sung WL, Davoodi J & Makoto Y (1994) Protein Sci 3 , 465–475, and Sabini E, Wilson KS, Danielsen S, Schülein M & Davies GJ (2001) Acta Crystallogr D57 , 1344–1347]. Therefore, the role of the aglycone subsites remains controversial despite their obvious contribution to catalysis.