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 Erik Mølager Christensen, Birthe B. Kragelund, Paul Dupree, Barry McCleary, Ole Hindsgaul, Maher Abou Hachem, Birte Svensson

Research output: Contribution to journalJournal articleResearchpeer-review

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; 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.
Original languageEnglish
JournalApplied Microbiology and Biotechnology
Volume100
Issue number14
Pages (from-to)6265-6277
Number of pages13
ISSN0175-7598
DOIs
Publication statusPublished - 2016

Keywords

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

Cite this

Wilkens, Casper ; Andersen, Susan ; Petersen, Bent O. ; Li, An ; Busse-Wicher, Marta ; Birch, Johnny ; Cockburn, Darrell ; Nakai, Hiroyuki ; Christensen, Hans Erik Mølager ; Kragelund, Birthe B. ; Dupree, Paul ; McCleary, Barry ; Hindsgaul, Ole ; Abou Hachem, Maher ; Svensson, Birte. / An efficient arabinoxylan-debranching α-l-arabinofuranosidase of family GH62 from Aspergillus nidulans contains a secondary carbohydrate binding site. In: Applied Microbiology and Biotechnology. 2016 ; Vol. 100, No. 14. pp. 6265-6277.
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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; 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 {\AA} 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",
author = "Casper Wilkens and Susan Andersen and Petersen, {Bent O.} and An Li and Marta Busse-Wicher and Johnny Birch and Darrell Cockburn and Hiroyuki Nakai and Christensen, {Hans Erik M{\o}lager} and Kragelund, {Birthe B.} and Paul Dupree and Barry McCleary and Ole Hindsgaul and {Abou Hachem}, Maher and Birte Svensson",
year = "2016",
doi = "10.1007/s00253-016-7417-8",
language = "English",
volume = "100",
pages = "6265--6277",
journal = "Applied Microbiology and Biotechnology",
issn = "0175-7598",
publisher = "Springer",
number = "14",

}

Wilkens, C, Andersen, S, Petersen, BO, Li, A, Busse-Wicher, M, Birch, J, Cockburn, D, Nakai, H, Christensen, HEM, Kragelund, BB, Dupree, P, McCleary, B, Hindsgaul, O, Abou Hachem, M & Svensson, B 2016, 'An efficient arabinoxylan-debranching α-l-arabinofuranosidase of family GH62 from Aspergillus nidulans contains a secondary carbohydrate binding site', Applied Microbiology and Biotechnology, vol. 100, no. 14, pp. 6265-6277. https://doi.org/10.1007/s00253-016-7417-8

An efficient arabinoxylan-debranching α-l-arabinofuranosidase of family GH62 from Aspergillus nidulans contains a secondary carbohydrate binding site. / Wilkens, Casper; Andersen, Susan; Petersen, Bent O.; Li, An; Busse-Wicher, Marta; Birch, Johnny; Cockburn, Darrell; Nakai, Hiroyuki; Christensen, Hans Erik Mølager; Kragelund, Birthe B.; Dupree, Paul; McCleary, Barry; Hindsgaul, Ole; Abou Hachem, Maher ; Svensson, Birte.

In: Applied Microbiology and Biotechnology, Vol. 100, No. 14, 2016, p. 6265-6277.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - An efficient arabinoxylan-debranching α-l-arabinofuranosidase of family GH62 from Aspergillus nidulans contains a secondary carbohydrate binding site

AU - Wilkens, Casper

AU - Andersen, Susan

AU - Petersen, Bent O.

AU - Li, An

AU - Busse-Wicher, Marta

AU - Birch, Johnny

AU - Cockburn, Darrell

AU - Nakai, Hiroyuki

AU - Christensen, Hans Erik Mølager

AU - Kragelund, Birthe B.

AU - Dupree, Paul

AU - McCleary, Barry

AU - Hindsgaul, Ole

AU - Abou Hachem, Maher

AU - Svensson, Birte

PY - 2016

Y1 - 2016

N2 - 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; 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.

AB - 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; 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.

KW - Glycoside hydrolase family 62

KW - Inverting mechanism

KW - Arabinoxylan

KW - Arabinoxylooligosaccharides

KW - Affinity gel electrophoresis

KW - Surface binding site

U2 - 10.1007/s00253-016-7417-8

DO - 10.1007/s00253-016-7417-8

M3 - Journal article

VL - 100

SP - 6265

EP - 6277

JO - Applied Microbiology and Biotechnology

JF - Applied Microbiology and Biotechnology

SN - 0175-7598

IS - 14

ER -