NMR Spectroscopic Analysis Reveals Extensive Binding Interactions of Complex Xyloglucan Oligosaccharides with the Cellvibrio japonicus Glycoside Hydrolase Family 31 α‐Xylosidase |
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Authors: | Dr Alba Silipo Johan Larsbrink Roberta Marchetti Prof Rosa Lanzetta Prof Harry Brumer Prof Antonio Molinaro |
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Institution: | 1. Dipartimento di Scienze Chimiche, Università di Napoli “Federico II”, Complesso Universitario Monte S. Angelo, Via Cintia 4, 80126 Napoli (Italy), Fax: (+39)?081‐674393;2. Division of Glycoscience, School of Biotechnology, Royal Institute of Technology (KTH), Albanova University Center, 106 91 Stockholm (Sweden);3. Michael Smith Laboratories and Department of Chemistry, University of British Columbia, 2185 East Mall, Vancouver, BC, V6T 1Z4 (Canada) |
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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. |
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Keywords: | α ‐xylosidases Cellvibrio japonicus molecular recognition mutagenesis NMR spectroscopy xyloglucan |
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