Journal article
An efficient arabinoxylan-debranching α-l-arabinofuranosidase of family GH62 from Aspergillus nidulans contains a secondary carbohydrate binding site
Department of Systems Biology, Technical University of Denmark1
University of Copenhagen2
Megazyme Ireland Ltd.3
Department of Chemical and Biochemical Engineering, Technical University of Denmark4
Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark5
Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark6
Carlsberg Research Center7
University of Cambridge8
Department of Chemistry, Technical University of Denmark9
Metalloprotein Chemistry and Engineering, Department of Chemistry, Technical University of Denmark10
Organic Chemistry, Department of Chemistry, Technical University of Denmark11
...and 1 moreAn α-l-arabinofuranosidase of GH62 from Aspergillus nidulans FGSC A4 (AnAbf62A-m2,3) has an unusually high activity towards wheat arabinoxylan (WAX) (67 U/mg; kcat = 178/s, Km = 4.90 mg/ml) and arabinoxylooligosaccharides (AXOS) with degrees of polymerisation (DP) 3–5 (37–80 U/mg), but about 50 times lower activity for sugar beet arabinan and 4-nitrophenyl-α-l-arabinofuranoside. α-1,2- and α-1,3-linked arabinofuranoses are released from monosubstituted, but not from disubstituted, xylose in WAX and different AXOS as demonstrated by NMR and polysaccharide analysis by carbohydrate gel electrophoresis (PACE).
Mutants of the predicted general acid (Glu188) and base (Asp28) catalysts, and the general acid pKa modulator (Asp136) lost 1700-, 165- and 130-fold activities for WAX. WAX, oat spelt xylan, birchwood xylan and barley β-glucan retarded migration of AnAbf62A-m2,3 in affinity electrophoresis (AE) although the latter two are neither substrates nor inhibitors.
Trp23 and Tyr44, situated about 30 Å from the catalytic site as seen in an AnAbf62A-m2,3 homology model generated using Streptomyces thermoviolaceus SthAbf62A as template, participate in carbohydrate binding. Compared to wild-type, W23A and W23A/Y44A mutants are less retarded in AE, maintain about 70 % activity towards WAX with K i of WAX substrate inhibition increasing 4–7-folds, but lost 77–96 % activity for the AXOS.
The Y44A single mutant had less effect, suggesting Trp23 is a key determinant. AnAbf62A-m2,3 seems to apply different polysaccharide-dependent binding modes, and Trp23 and Tyr44 belong to a putative surface binding site which is situated at a distance of the active site and has to be occupied to achieve full activity.
Language: | English |
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Publisher: | Springer Berlin Heidelberg |
Year: | 2016 |
Pages: | 6265-6277 |
ISSN: | 14320614 and 01757598 |
Types: | Journal article |
DOI: | 10.1007/s00253-016-7417-8 |
ORCIDs: | Wilkens, Casper , Christensen, Hans Erik Mølager , Abou Hachem, Maher , Svensson, Birte and 0000-0002-7454-1761 |
Affinity gel electrophoresis Arabinoxylan Arabinoxylooligosaccharides Glycoside hydrolase family 62 Inverting mechanism Surface binding site
4-nitrophenyl-alpha-L-arabinofuranoside Arabinose Aspergillus nidulans Binding Sites Biotechnology Catalytic Domain Cloning, Molecular Fungal Proteins Gene Expression Regulation, Fungal Glycoside Hydrolases Life Sciences Microbial Genetics and Genomics Microbiology Phylogeny Pichia Polysaccharides Protein Conformation SC3 Streptomyces Substrate Specificity Triticum Xylans Xylose alpha-N-arabinofuranosidase araban arabinoxylan beta-Glucans