title: An efficient arabinoxylan-debranching α-L-arabinofuranosidase of family GH62 from Aspergillus nidulans contains a secondary carbohydrate binding site.
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 (Glu(188)) and base (Asp(28)) catalysts, and the general acid pK a modulator (Asp(136)) 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. Trp(23) and Tyr(44), 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 Trp(23) is a key determinant. AnAbf62A-m2,3 seems to apply different polysaccharide-dependent binding modes, and Trp(23) and Tyr(44) 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.
authors.affiliation: Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Elektrovej, Building 375, 2800, Kgs. Lyngby, Denmark.|Department of Chemical and Biochemical Engineering, Technical University of Denmark, Søltofts Plads, Building 227, 2800, Kgs. Lyngby, Denmark.
authors.forename: Casper
authors.initials: C
authors.lastname: Wilkens
authors.affiliation: Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Elektrovej, Building 375, 2800, Kgs. Lyngby, Denmark.
authors.forename: Susan
authors.initials: S
authors.lastname: Andersen
authors.affiliation: Carbohydrate Chemistry Group, Carlsberg Laboratory, Gamle Carlsberg Vej 10, 1799, Copenhagen V, Denmark.|Biophysics and Biotechnology, Novo Nordisk A/S, Novo Nordisk Park, 2760, Måløv, Denmark.
authors.forename: Bent O
authors.initials: BO
authors.lastname: Petersen
authors.affiliation: Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QW, UK.
authors.forename: An
authors.initials: A
authors.lastname: Li
authors.affiliation: Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QW, UK.
authors.forename: Marta
authors.initials: M
authors.lastname: Busse-Wicher
authors.affiliation: Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Elektrovej, Building 375, 2800, Kgs. Lyngby, Denmark.
authors.forename: Johnny
authors.initials: J
authors.lastname: Birch
authors.affiliation: Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Elektrovej, Building 375, 2800, Kgs. Lyngby, Denmark.|Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI, MI 48109, USA.
authors.forename: Darrell
authors.initials: D
authors.lastname: Cockburn
authors.affiliation: Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Elektrovej, Building 375, 2800, Kgs. Lyngby, Denmark.|Graduate School of Science and Technology, Niigata University, 8050 Ikarashi, Nishi-ku, Niigata, 950-2181, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Nakai
authors.affiliation: Metalloprotein Chemistry and Engineering, Department of Chemistry, Technical University of Denmark, Kemitorvet, Building 207, 2800, Kgs. Lyngby, Denmark.
authors.forename: Hans E M
authors.initials: HEM
authors.lastname: Christensen
authors.affiliation: Structural Biology and NMR Laboratory, Department of Biology, University of Copenhagen, Ole Maaloes Vej 5, 2200, Copenhagen N, Denmark.
authors.forename: Birthe B
authors.initials: BB
authors.lastname: Kragelund
authors.affiliation: Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QW, UK.
authors.forename: Paul
authors.initials: P
authors.lastname: Dupree
authors.affiliation: Megazyme International, Bray Business Park, Bray, Co., Wicklow, Ireland.
authors.forename: Barry
authors.initials: B
authors.lastname: McCleary
authors.affiliation: Carbohydrate Chemistry Group, Carlsberg Laboratory, Gamle Carlsberg Vej 10, 1799, Copenhagen V, Denmark.
authors.forename: Ole
authors.initials: O
authors.lastname: Hindsgaul
authors.affiliation: Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Elektrovej, Building 375, 2800, Kgs. Lyngby, Denmark.
authors.forename: Maher Abou
authors.initials: MA
authors.lastname: Hachem
authors.affiliation: Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Elektrovej, Building 375, 2800, Kgs. Lyngby, Denmark. bis@bio.dtu.dk.
authors.forename: Birte
authors.initials: B
authors.lastname: Svensson
mesh_terms: D001089:Arabinose / Q000031:analogs & derivatives / Q000737:chemistry; D001233:Aspergillus nidulans / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D020134:Catalytic Domain; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D006026:Glycoside Hydrolases / Q000737:chemistry*; D010802:Phylogeny; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry; D011487:Protein Conformation; D013302:Streptomyces / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014908:Triticum / Q000737:chemistry; D014990:Xylans / Q000737:chemistry*; D014994:Xylose / Q000737:chemistry; D047071:beta-Glucans / Q000737:chemistry
chemical_list: D005656:Fungal Proteins; D011134:Polysaccharides; D014990:Xylans; D047071:beta-Glucans; C030080:araban; C017567:4-nitrophenyl-alpha-L-arabinofuranoside; C085118:arabinoxylan; D014994:Xylose; D001089:Arabinose; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase
keywords: Affinity gel electrophoresis; Arabinoxylan; Arabinoxylooligosaccharides; Glycoside hydrolase family 62; Inverting mechanism; Surface binding site