title: Elucidation of the molecular basis for arabinoxylan-debranching activity of a thermostable family GH62 α-l-arabinofuranosidase from Streptomyces thermoviolaceus.
abstract: Xylan-debranching enzymes facilitate the complete hydrolysis of xylan and can be used to alter xylan chemistry. Here, the family GH62 α-l-arabinofuranosidase from Streptomyces thermoviolaceus (SthAbf62A) was shown to have a half-life of 60 min at 60°C and the ability to cleave α-1,3 l-arabinofuranose (l-Araf) from singly substituted xylopyranosyl (Xylp) backbone residues in wheat arabinoxylan; low levels of activity on arabinan as well as 4-nitrophenyl α-l-arabinofuranoside were also detected. After selective removal of α-1,3 l-Araf substituents from disubstituted Xylp residues present in wheat arabinoxylan, SthAbf62A could also cleave the remaining α-1,2 l-Araf substituents, confirming the ability of SthAbf62A to remove α-l-Araf residues that are (1→2) and (1→3) linked to monosubstituted β-d-Xylp sugars. Three-dimensional structures of SthAbf62A and its complex with xylotetraose and l-arabinose confirmed a five-bladed β-propeller fold and revealed a molecular Velcro in blade V between the β1 and β21 strands, a disulfide bond between Cys27 and Cys297, and a calcium ion coordinated in the central channel of the fold. The enzyme-arabinose complex structure further revealed a narrow and seemingly rigid l-arabinose binding pocket situated at the center of one side of the β propeller, which stabilized the arabinofuranosyl substituent through several hydrogen-bonding and hydrophobic interactions. The predicted catalytic amino acids were oriented toward this binding pocket, and the catalytic essentiality of Asp53 and Glu213 was confirmed by site-specific mutagenesis. Complex structures with xylotetraose revealed a shallow cleft for xylan backbone binding that is open at both ends and comprises multiple binding subsites above and flanking the l-arabinose binding pocket.
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Weijun
authors.initials: W
authors.lastname: Wang
authors.affiliation: Department of Biotechnology and Chemical Technology, Aalto University, Espoo, Finland.
authors.forename: Galina
authors.initials: G
authors.lastname: Mai-Gisondi
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Stogios
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Amrit
authors.initials: A
authors.lastname: Kaur
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Xiaohui
authors.initials: X
authors.lastname: Xu
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Hong
authors.initials: H
authors.lastname: Cui
authors.affiliation: Department of Biotechnology and Chemical Technology, Aalto University, Espoo, Finland.
authors.forename: Ossi
authors.initials: O
authors.lastname: Turunen
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Alexei
authors.initials: A
authors.lastname: Savchenko
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada Department of Biotechnology and Chemical Technology, Aalto University, Espoo, Finland emma.master@utoronto.ca.
authors.forename: Emma R
authors.initials: ER
authors.lastname: Master
mesh_terms: D001665:Binding Sites; D018360:Crystallography, X-Ray; D004252:DNA Mutational Analysis; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors; D014990:Xylans / Q000378:metabolism*
chemical_list: D014990:Xylans; C085118:arabinoxylan; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase