National Center for
4O8P: Crystal Structure Of Stharaf62a, A Gh62 Family Alpha-l- Arabinofuranosidase From Streptomyces Thermoviolaceus, Bound To Xylotetraose
Biochemical and structural characterization of a thermostable family GH62 alpha-l-arabinofuranosidase from Streptomyces thermoviolaceus to elucidate the molecular basis for activity towards arabinoxylan
Appl. Environ. Microbiol. (2014)
Xylan debranching enzymes facilitate the complete hydrolysis of xylan and can be used to alter xylan chemistry. Herein, the GH62 family alpha-l-arabinofuranosidase from Streptomyces thermoviolaceus (SthAbf62A) was shown to have a half-life of 60 min at 60 degrees C, and ability to cleave alpha-1,3 l-arabinofuranose (l-Araf ) from singly-substituted xylopyranosyl (Xylp) backbone residues in wheat arabinoxylan; low activity towards arabinan as well as 4-nitrophenyl alpha-l-arabinofuranoside was also detected. After selectively removing alpha-1,3 l-Araf substituents from di-substituted Xylp residues present in wheat arabinoxylan, SthAbf62A could also cleave the remaining alpha-1,2 l-Araf substituents, confirming the ability of SthAbf62A to remove alpha-l-Araf residues that are (1-->2) and (1-->3) linked to mono-substituted beta-d-Xylp sugars. Three-dimensional structures of SthAbf62A and its complex with xylotetraose and l-arabinose confirmed a five-bladed beta-propeller fold and revealed a molecular Velcro in blade V between the beta1 and beta21 strands, a disulfide bond between Cys 27 and Cys 297, 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 beta propeller, which stabilized the arabinofuranosyl substituent through several hydrogen-bonding and hydrophobic interactions. The predicted catalytic amino acids were oriented towards 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 which is open at both ends and comprises multiple binding subsites above and flanking the l-arabinose binding pocket.