title: Both substrate hydrolysis and secondary substrate binding determine xylanase mobility as assessed by FRAP.
abstract: Xylanases (EC 3.2.1.8) are enzymes that can hydrolyze the xylan backbone internally. Therefore, they are important for biomass breakdown and they are also often added in various biotechnological applications. In this study, the relationship between their substrate binding affinity and hydrolysis, on the one hand, and their movement over natural substrates, on the other hand, was investigated. Fluorescence recovery after photobleaching (FRAP) experiments using different Bacillus subtilis xylanase A (XBS) mutants were conducted on water-unextractable wheat flour arabinoxylan (WU-AX) and insoluble oat spelt xylan (OSX). To assess the importance of substrate hydrolysis, FRAP of a catalytically inactive mutant was compared to that of the wild-type enzyme. For the wild-type enzyme, substrate binding and a complete recovery of fluorescence after photobleaching was observed on both substrates. For the inactive mutant, however, substrate binding but no fluorescence recovery was observed on WU-AX, while very slow recovery was observed on OSX. Furthermore, the importance of substrate binding to a secondary xylan binding site (SBS) for enzyme mobility was studied by testing two mutants with a modified SBS (N54W-N141Q and G56A-T183A-W185A) that showed different behavior on the tested substrates. On OSX, the two modified enzymes both showed higher mobility than the wild-type enzyme. On WU-AX, in contrast, the N54W-N141Q mutant displayed a lower mobility than the wild-type enzyme, while the G56A-T183A-W185A mutant showed higher mobility. The results clearly demonstrate that both substrate hydrolysis and substrate targeting are key factors for XBS mobility.
authors.affiliation: Laboratory of Food Chemistry and Biochemistry & Leuven Food Science and Nutrition Research Centre (LFoRCe), Katholieke Universiteit Leuven, Leuven, Belgium.
authors.forename: Sven
authors.initials: S
authors.lastname: Cuyvers
authors.forename: Jelle
authors.initials: J
authors.lastname: Hendrix
authors.forename: Emmie
authors.initials: E
authors.lastname: Dornez
authors.forename: Yves
authors.initials: Y
authors.lastname: Engelborghs
authors.forename: Jan A
authors.initials: JA
authors.lastname: Delcour
authors.forename: Christophe M
authors.initials: CM
authors.lastname: Courtin
mesh_terms: D019943:Amino Acid Substitution; D001412:Bacillus subtilis / Q000201:enzymology; D001665:Binding Sites; D055162:Biocatalysis; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods*; D006868:Hydrolysis; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014990:Xylans / Q000737:chemistry / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D014990:Xylans; C085118:arabinoxylan; D043364:Endo-1,4-beta Xylanases