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

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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.

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Acknowledgments

Mette Pries is thanked for technical assistance and Anne Blicher for amino acid analysis. The 800 MHz NMR spectra were recorded at the Danish National Instrument Centre for NMR spectroscopy of Biological Macromolecules at the Carlsberg Laboratory. Maja Tenkanen (University of Helsinki) is thanked for doubly substituted AXOS.

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Author notes

  1. Casper Wilkens

    Present address: Department of Chemical and Biochemical Engineering, Technical University of Denmark, Søltofts Plads, Building 227, 2800, Kgs. Lyngby, Denmark

  2. Bent O. Petersen

    Present address: Biophysics and Biotechnology, Novo Nordisk A/S, Novo Nordisk Park, 2760, Måløv, Denmark

  3. Darrell Cockburn

    Present address: Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI, MI 48109, USA

  4. Hiroyuki Nakai

    Present address: Graduate School of Science and Technology, Niigata University, 8050 Ikarashi, Nishi-ku, Niigata, 950-2181, Japan

Authors and Affiliations

  1. Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Elektrovej, Building 375, 2800, Kgs. Lyngby, Denmark

    Casper Wilkens, Susan Andersen, Johnny Birch, Darrell Cockburn, Hiroyuki Nakai, Maher Abou Hachem & Birte Svensson

  2. Carbohydrate Chemistry Group, Carlsberg Laboratory, Gamle Carlsberg Vej 10, 1799, Copenhagen V, Denmark

    Bent O. Petersen & Ole Hindsgaul

  3. Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QW, UK

    An Li, Marta Busse-Wicher & Paul Dupree

  4. Metalloprotein Chemistry and Engineering, Department of Chemistry, Technical University of Denmark, Kemitorvet, Building 207, 2800, Kgs. Lyngby, Denmark

    Hans E. M. Christensen

  5. Structural Biology and NMR Laboratory, Department of Biology, University of Copenhagen, Ole Maaloes Vej 5, 2200, Copenhagen N, Denmark

    Birthe B. Kragelund

  6. Megazyme International, Bray Business Park, Bray, Co., Wicklow, Ireland

    Barry McCleary

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  1. Casper Wilkens
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Correspondence to Birte Svensson.

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This article does not contain any studies with human participants or animals performed by any of the authors.

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This work is supported by the Danish Council for Independent Research|Natural Sciences (FNU) [grant number 09-072151], by 1/3 PhD fellowship from the Technical University of Denmark (to CW) and by a Hans Christian Ørsted postdoctoral fellowship from DTU (to DC).

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Barry McCleary is the CEO and founder of Megazyme International.

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Wilkens, C., Andersen, S., Petersen, B.O. et al. An efficient arabinoxylan-debranching α-l-arabinofuranosidase of family GH62 from Aspergillus nidulans contains a secondary carbohydrate binding site. Appl Microbiol Biotechnol 100, 6265–6277 (2016). https://doi.org/10.1007/s00253-016-7417-8

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