Open AccessFunctional and structural diversity in GH 62 α‐ L ‐arabinofuranosidases from the thermophilic fungus S cytalidium thermophilum
Author(s) -
Kaur Amrit Pal,
Nocek Boguslaw P.,
Xu Xiaohui,
Lowden Michael J.,
Leyva Juan Francisco,
Stogios Peter J.,
Cui Hong,
Di Leo Rosa,
Powlowski Justin,
Tsang Adrian,
Savchenko Alexei
Publication year - 2015
Publication title -
microbial biotechnology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.287
H-Index - 74
ISSN - 1751-7915
DOI - 10.1111/1751-7915.12168
Subject(s) - glycoside hydrolase , catalytic triad , biochemistry , biology , escherichia coli , thermophile , gene , chemistry , genetics , enzyme , peptide sequence
Summary The genome of the thermophilic fungus S cytalidium thermophilum (strain CBS 625.91) harbours a wide range of genes involved in carbohydrate degradation, including three genes, abf62A , abf62B and abf62C , predicted to encode glycoside hydrolase family 62 ( GH 62) enzymes. Transcriptome analysis showed that only abf62A and abf62C are actively expressed during growth on diverse substrates including straws from barley, alfalfa, triticale and canola. The abf62A and abf62C genes were expressed in E scherichia coli and the resulting recombinant proteins were characterized. Calcium‐free crystal structures of Abf62C in apo and xylotriose bound forms were determined to 1.23 and 1.48 Å resolution respectively. Site‐directed mutagenesis confirmed A sp55, A sp171 and G lu230 as catalytic triad residues, and revealed the critical role of non‐catalytic residues A sp194, T rp229 and T yr338 in positioning the scissile α‐ L ‐arabinofuranoside bond at the catalytic site. Further, the +2 R substrate‐binding site residues T yr168 and A sn339, as well as the +2 NR residue T yr226, are involved in accommodating long‐chain xylan polymers. Overall, our structural and functional analysis highlights characteristic differences between Abf62A and Abf62C , which represent divergent subgroups in the GH 62 family.