Elucidation of the molecular basis for arabinoxylan-debranching activity of a thermostable family GH62 ?-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 ?-l-arabinofuranosidase from Streptomyces thermoviolaceus (SthAbf62A) was shown to have a half-life of 60 min at 60°C and the ability to cleave ?-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 ?-l-arabinofuranoside were also detected. After selective removal of ?-1,3 l-Araf substituents from disubstituted Xylp residues present in wheat arabinoxylan, SthAbf62A could also cleave the remaining ?-1,2 l-Araf substituents, confirming the ability of SthAbf62A to remove ?-l-Araf residues that are (1?2) and (1?3) linked to monosubstituted ?-d-Xylp sugars. Three-dimensional structures of SthAbf62A and its complex with xylotetraose and l-arabinose confirmed a five-bladed ?-propeller fold and revealed a molecular Velcro in blade V between the ?1 and ?21 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 ? 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.

SUBMITTER: Wang W 

PROVIDER: S-EPMC4136092 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature