title: NMR spectroscopic analysis reveals extensive binding interactions of complex xyloglucan oligosaccharides with the Cellvibrio japonicus glycoside hydrolase family 31 α-xylosidase.
abstract: The study of the interaction of glycoside hydrolases with their substrates is fundamental to diverse applications in medicine, food and feed production, and biomass-resource utilization. Recent molecular modeling of the α-xylosidase CjXyl31A from the soil saprophyte Cellvibrio japonicus, together with protein crystallography and enzyme-kinetic analysis, has suggested that an appended PA14 protein domain, unique among glycoside hydrolase family 31 members, may confer specificity for large oligosaccharide fragments of the ubiquitous plant polysaccharide xyloglucan (J. Larsbrink, A. Izumi, F.M. Ibatullin, A. Nakhai, H.J. Gilbert, G.J. Davies, H. Brumer, Biochem. J. 2011, 436, 567-580). In the present study, a combination of NMR spectroscopic techniques, including saturation transfer difference (STD) and transfer NOE (TR-NOE) spectroscopy, was used to reveal extensive interactions between CjXyl31A active-site variants and xyloglucan hexa- and heptasaccharides. The data specifically indicate that the enzyme recognizes the entire cello-tetraosyl backbone of the substrate and product in positive enzyme subsites and makes further significant interactions with internal pendant α-(1→6)-linked xylosyl units. As such, the present analysis provides an important rationalization of previous kinetic data on CjXyl31A and unique insight into the role of the PA14 domain, which was not otherwise obtainable by protein crystallography.
authors.affiliation: Dipartimento di Scienze Chimiche, Università di Napoli Federico II, Complesso Universitario Monte S. Angelo, Via Cintia 4, 80126 Napoli, Italy. silipo@unina.it
authors.forename: Alba
authors.initials: A
authors.lastname: Silipo
authors.forename: Johan
authors.initials: J
authors.lastname: Larsbrink
authors.forename: Roberta
authors.initials: R
authors.lastname: Marchetti
authors.forename: Rosa
authors.initials: R
authors.lastname: Lanzetta
authors.forename: Harry
authors.initials: H
authors.lastname: Brumer
authors.forename: Antonio
authors.initials: A
authors.lastname: Molinaro
mesh_terms: D001665:Binding Sites; D020134:Catalytic Domain; D020633:Cellvibrio / Q000201:enzymology*; D005936:Glucans / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D062105:Molecular Docking Simulation; D016296:Mutagenesis; D009844:Oligosaccharides / Q000737:chemistry*; D013237:Stereoisomerism; D014990:Xylans / Q000737:chemistry*; D014995:Xylosidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D005936:Glucans; D009844:Oligosaccharides; D014990:Xylans; C029353:xyloglucan; D014995:Xylosidases