NDLI: An efficient arabinoxylan-debranching α-l-arabinofuranosidase of family GH62 from Aspergillus nidulans contains a secondary carbohydrate binding site

Content Provider	Springer Nature Link
Author	Wilkens, Casper Andersen, Susan Petersen, Bent O. Li, An Busse Wicher, Marta Birch, Johnny Cockburn, Darrell Nakai, Hiroyuki Christensen, Hans E. M. Kragelund, Birthe B. Dupree, Paul McCleary, Barry Hindsgaul, Ole Hachem, Maher Abou Svensson, Birte
Copyright Year	2016
Abstract	An 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; k cat = 178/s, K m = 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 pK a 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.
Starting Page	6265
Ending Page	6277
Page Count	13
File Format	PDF
ISSN	01757598
Journal	Applied Microbiology and Biotechnology
Volume Number	100
Issue Number	14
e-ISSN	14320614
Language	English
Publisher	Springer Berlin Heidelberg
Publisher Date	2016-03-05
Publisher Place	Berlin, Heidelberg
Access Restriction	Subscribed
Subject Keyword	Glycoside hydrolase family 62 Inverting mechanism Arabinoxylan Arabinoxylooligosaccharides Affinity gel electrophoresis Surface binding site Microbiology Microbial Genetics and Genomics Biotechnology
Content Type	Text
Resource Type	Article
Subject	Medicine Applied Microbiology and Biotechnology Biotechnology

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