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 Brandt Kragelund; Paul Dupree; Barry McCleary; Ole Hindsgaul; Maher Abou Hachem; Birte Svensson

doi: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

Casper Wilkens, Susan Andersen, Bent O. Petersen, An Li, Marta Busse-Wicher, Johnny Birch, Darrell Cockburn, Hiroyuki Nakai, Hans Erik Mølager Christensen, Birthe Brandt Kragelund, Paul Dupree, Barry McCleary, Ole Hindsgaul, Maher Abou Hachem, Birte Svensson

Biomolecular Sciences

15 Citations (Scopus)

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.

Original language	English
Journal	Applied Microbiology and Biotechnology
Volume	100
Issue number	14
Pages (from-to)	6265-6277
Number of pages	13
ISSN	0175-7598
DOIs	https://doi.org/10.1007/s00253-016-7417-8
Publication status	Published - 1 Jul 2016

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Wilkens, C., Andersen, S., Petersen, B. O., Li, A., Busse-Wicher, M., Birch, J., Cockburn, D., Nakai, H., Christensen, H. E. M., Kragelund, B. B., 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, 100(14), 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. et al.
In: Applied Microbiology and Biotechnology, Vol. 100, No. 14, 01.07.2016, p. 6265-6277.

Research output: Contribution to journal › Journal article › Research › peer-review

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

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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; 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 {\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 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.",

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 Brandt} and Paul Dupree and Barry McCleary and Ole Hindsgaul and {Abou Hachem}, Maher and Birte Svensson",

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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 Brandt

AU - Dupree, Paul

AU - McCleary, Barry

AU - Hindsgaul, Ole

AU - Abou Hachem, Maher

AU - Svensson, Birte

PY - 2016/7/1

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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; 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.

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; 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.

U2 - 10.1007/s00253-016-7417-8

DO - 10.1007/s00253-016-7417-8

M3 - Journal article

C2 - 26946172

SN - 0175-7598

VL - 100

SP - 6265

EP - 6277

JO - Applied Microbiology and Biotechnology

JF - Applied Microbiology and Biotechnology

IS - 14

ER -

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

Abstract

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