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Applied Microbiology and Biotechnology

, Volume 100, Issue 14, pp 6265–6277 | Cite as

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

  • Casper Wilkens
  • Susan Andersen
  • Bent O. Petersen
  • An Li
  • Marta Busse-Wicher
  • Johnny Birch
  • Darrell Cockburn
  • Hiroyuki Nakai
  • Hans E. M. Christensen
  • Birthe B. Kragelund
  • Paul Dupree
  • Barry McCleary
  • Ole Hindsgaul
  • Maher Abou Hachem
  • Birte SvenssonEmail author
Biotechnologically relevant enzymes and proteins

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.

Keywords

Glycoside hydrolase family 62 Inverting mechanism Arabinoxylan Arabinoxylooligosaccharides Affinity gel electrophoresis Surface binding site 

Notes

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.

Compliance with ethical standards

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

Funding

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).

Conflict of interest

Barry McCleary is the CEO and founder of Megazyme International.

Supplementary material

253_2016_7417_MOESM1_ESM.pdf (924 kb)
ESM 1 (PDF 923 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Casper Wilkens
    • 1
    • 2
  • Susan Andersen
    • 1
  • Bent O. Petersen
    • 3
    • 4
  • An Li
    • 5
  • Marta Busse-Wicher
    • 5
  • Johnny Birch
    • 1
  • Darrell Cockburn
    • 1
    • 6
  • Hiroyuki Nakai
    • 1
    • 7
  • Hans E. M. Christensen
    • 8
  • Birthe B. Kragelund
    • 9
  • Paul Dupree
    • 5
  • Barry McCleary
    • 10
  • Ole Hindsgaul
    • 3
  • Maher Abou Hachem
    • 1
  • Birte Svensson
    • 1
    Email author
  1. 1.Enzyme and Protein Chemistry, Department of Systems BiologyTechnical University of DenmarkKgs. LyngbyDenmark
  2. 2.Department of Chemical and Biochemical EngineeringTechnical University of DenmarkKgs. LyngbyDenmark
  3. 3.Carbohydrate Chemistry Group, Carlsberg LaboratoryCopenhagen VDenmark
  4. 4.Biophysics and BiotechnologyMåløvDenmark
  5. 5.Department of BiochemistryUniversity of CambridgeCambridgeUK
  6. 6.Department of Microbiology and ImmunologyUniversity of Michigan Medical SchoolAnn ArborUSA
  7. 7.Graduate School of Science and TechnologyNiigata UniversityNiigataJapan
  8. 8.Metalloprotein Chemistry and Engineering, Department of ChemistryTechnical University of DenmarkKgs. LyngbyDenmark
  9. 9.Structural Biology and NMR Laboratory, Department of BiologyUniversity of CopenhagenCopenhagen NDenmark
  10. 10.Megazyme InternationalWicklowIreland

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