A carbohydrate-binding family 48 module enables feruloyl esterase action on polymeric arabinoxylan

Jesper Holck, Folmer Fredslund, Marie Sofie Møller, Jesper Brask, Kristian B. R. M. Krogh, Lene Lange, Ditte Hededam Welner, Birte Svensson, Anne S. Meyer, Casper Wilkens*

*Corresponding author for this work

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Abstract

Feruloyl esterases (EC 3.1.1.73), belonging to carbohydrate esterase family 1 (CE1), hydrolyze ester bonds between ferulic acid (FA) and arabinose moieties in arabinoxylans. Recently, some CE1 enzymes identified in metagenomics studies have been predicted to contain a family 48 carbohydrate-binding module (CBM48), a CBM family associated with starch binding. Two of these CE1s, wtsFae1A and wtsFae1B isolated from wastewater treatment surplus sludge, have a cognate CBM48 domain and are feruloyl esterases, and wtsFae1A binds arabinoxylan. Here, we show that wtsFae1B also binds to arabinoxylan and that neither binds starch. Surface plasmon resonance analysis revealed that wtsFae1B's Kd for xylohexaose is 14.8 μM and that it does not bind to starch mimics, β-cyclodextrin, or maltohexaose. Interestingly, in the absence of CBM48 domains, the CE1 regions from wtsFae1A and wtsFae1B did not bind arabinoxylan and were also unable to catalyze FA release from arabinoxylan. Pretreatment with a β-D-1,4-xylanase did enable CE1 domain-mediated FA release from arabinoxylan in the absence of CBM48, indicating that CBM48 is essential for the CE1 activity on the polysaccharide. Crystal structures of wtsFae1A (at 1.63 Å resolution) and wtsFae1B (1.98 Å) revealed that both are folded proteins comprising structurally conserved hydrogen bonds that lock the CBM48 position relative to that of the CE1 domain. wtsFae1A docking indicated that both enzymes accommodate the arabinoxylan backbone in a cleft at the CE1-CBM48 domain interface. Binding at this cleft appears to enable CE1 activities on polymeric arabinoxylan, illustrating an unexpected and crucial role of CBM48 domains for accommodating arabinoxylan.
Original languageEnglish
Article numberjbc.RA119.009523
JournalJournal of Biological Chemistry
Volume294
Issue number46
Pages (from-to)17339-17353
Number of pages16
ISSN0021-9258
DOIs
Publication statusPublished - 2019

Keywords

  • Arabinoxylan
  • Carbonydrate binding module
  • Carbohydrate esterase family 1
  • Crystal structure
  • Enzyme catalysis
  • Enzyme mechanism
  • Ferulic acid esterase
  • Molecular docking
  • Molecular dynamics
  • Structure-function

Cite this

@article{17616b7203df4dafa46fdab945081b20,
title = "A carbohydrate-binding family 48 module enables feruloyl esterase action on polymeric arabinoxylan",
abstract = "Feruloyl esterases (EC 3.1.1.73), belonging to carbohydrate esterase family 1 (CE1), hydrolyze ester bonds between ferulic acid (FA) and arabinose moieties in arabinoxylans. Recently, some CE1 enzymes identified in metagenomics studies have been predicted to contain a family 48 carbohydrate-binding module (CBM48), a CBM family associated with starch binding. Two of these CE1s, wtsFae1A and wtsFae1B isolated from wastewater treatment surplus sludge, have a cognate CBM48 domain and are feruloyl esterases, and wtsFae1A binds arabinoxylan. Here, we show that wtsFae1B also binds to arabinoxylan and that neither binds starch. Surface plasmon resonance analysis revealed that wtsFae1B's Kd for xylohexaose is 14.8 μM and that it does not bind to starch mimics, β-cyclodextrin, or maltohexaose. Interestingly, in the absence of CBM48 domains, the CE1 regions from wtsFae1A and wtsFae1B did not bind arabinoxylan and were also unable to catalyze FA release from arabinoxylan. Pretreatment with a β-D-1,4-xylanase did enable CE1 domain-mediated FA release from arabinoxylan in the absence of CBM48, indicating that CBM48 is essential for the CE1 activity on the polysaccharide. Crystal structures of wtsFae1A (at 1.63 {\AA} resolution) and wtsFae1B (1.98 {\AA}) revealed that both are folded proteins comprising structurally conserved hydrogen bonds that lock the CBM48 position relative to that of the CE1 domain. wtsFae1A docking indicated that both enzymes accommodate the arabinoxylan backbone in a cleft at the CE1-CBM48 domain interface. Binding at this cleft appears to enable CE1 activities on polymeric arabinoxylan, illustrating an unexpected and crucial role of CBM48 domains for accommodating arabinoxylan.",
keywords = "Arabinoxylan, Carbonydrate binding module, Carbohydrate esterase family 1, Crystal structure, Enzyme catalysis, Enzyme mechanism, Ferulic acid esterase, Molecular docking, Molecular dynamics, Structure-function",
author = "Jesper Holck and Folmer Fredslund and M{\o}ller, {Marie Sofie} and Jesper Brask and Krogh, {Kristian B. R. M.} and Lene Lange and Welner, {Ditte Hededam} and Birte Svensson and Meyer, {Anne S.} and Casper Wilkens",
year = "2019",
doi = "10.1074/jbc.RA119.009523",
language = "English",
volume = "294",
pages = "17339--17353",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology, Inc.",
number = "46",

}

A carbohydrate-binding family 48 module enables feruloyl esterase action on polymeric arabinoxylan. / Holck, Jesper; Fredslund, Folmer; Møller, Marie Sofie; Brask, Jesper; Krogh, Kristian B. R. M.; Lange, Lene; Welner, Ditte Hededam; Svensson, Birte; Meyer, Anne S.; Wilkens, Casper.

In: Journal of Biological Chemistry, Vol. 294, No. 46, jbc.RA119.009523, 2019, p. 17339-17353.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - A carbohydrate-binding family 48 module enables feruloyl esterase action on polymeric arabinoxylan

AU - Holck, Jesper

AU - Fredslund, Folmer

AU - Møller, Marie Sofie

AU - Brask, Jesper

AU - Krogh, Kristian B. R. M.

AU - Lange, Lene

AU - Welner, Ditte Hededam

AU - Svensson, Birte

AU - Meyer, Anne S.

AU - Wilkens, Casper

PY - 2019

Y1 - 2019

N2 - Feruloyl esterases (EC 3.1.1.73), belonging to carbohydrate esterase family 1 (CE1), hydrolyze ester bonds between ferulic acid (FA) and arabinose moieties in arabinoxylans. Recently, some CE1 enzymes identified in metagenomics studies have been predicted to contain a family 48 carbohydrate-binding module (CBM48), a CBM family associated with starch binding. Two of these CE1s, wtsFae1A and wtsFae1B isolated from wastewater treatment surplus sludge, have a cognate CBM48 domain and are feruloyl esterases, and wtsFae1A binds arabinoxylan. Here, we show that wtsFae1B also binds to arabinoxylan and that neither binds starch. Surface plasmon resonance analysis revealed that wtsFae1B's Kd for xylohexaose is 14.8 μM and that it does not bind to starch mimics, β-cyclodextrin, or maltohexaose. Interestingly, in the absence of CBM48 domains, the CE1 regions from wtsFae1A and wtsFae1B did not bind arabinoxylan and were also unable to catalyze FA release from arabinoxylan. Pretreatment with a β-D-1,4-xylanase did enable CE1 domain-mediated FA release from arabinoxylan in the absence of CBM48, indicating that CBM48 is essential for the CE1 activity on the polysaccharide. Crystal structures of wtsFae1A (at 1.63 Å resolution) and wtsFae1B (1.98 Å) revealed that both are folded proteins comprising structurally conserved hydrogen bonds that lock the CBM48 position relative to that of the CE1 domain. wtsFae1A docking indicated that both enzymes accommodate the arabinoxylan backbone in a cleft at the CE1-CBM48 domain interface. Binding at this cleft appears to enable CE1 activities on polymeric arabinoxylan, illustrating an unexpected and crucial role of CBM48 domains for accommodating arabinoxylan.

AB - Feruloyl esterases (EC 3.1.1.73), belonging to carbohydrate esterase family 1 (CE1), hydrolyze ester bonds between ferulic acid (FA) and arabinose moieties in arabinoxylans. Recently, some CE1 enzymes identified in metagenomics studies have been predicted to contain a family 48 carbohydrate-binding module (CBM48), a CBM family associated with starch binding. Two of these CE1s, wtsFae1A and wtsFae1B isolated from wastewater treatment surplus sludge, have a cognate CBM48 domain and are feruloyl esterases, and wtsFae1A binds arabinoxylan. Here, we show that wtsFae1B also binds to arabinoxylan and that neither binds starch. Surface plasmon resonance analysis revealed that wtsFae1B's Kd for xylohexaose is 14.8 μM and that it does not bind to starch mimics, β-cyclodextrin, or maltohexaose. Interestingly, in the absence of CBM48 domains, the CE1 regions from wtsFae1A and wtsFae1B did not bind arabinoxylan and were also unable to catalyze FA release from arabinoxylan. Pretreatment with a β-D-1,4-xylanase did enable CE1 domain-mediated FA release from arabinoxylan in the absence of CBM48, indicating that CBM48 is essential for the CE1 activity on the polysaccharide. Crystal structures of wtsFae1A (at 1.63 Å resolution) and wtsFae1B (1.98 Å) revealed that both are folded proteins comprising structurally conserved hydrogen bonds that lock the CBM48 position relative to that of the CE1 domain. wtsFae1A docking indicated that both enzymes accommodate the arabinoxylan backbone in a cleft at the CE1-CBM48 domain interface. Binding at this cleft appears to enable CE1 activities on polymeric arabinoxylan, illustrating an unexpected and crucial role of CBM48 domains for accommodating arabinoxylan.

KW - Arabinoxylan

KW - Carbonydrate binding module

KW - Carbohydrate esterase family 1

KW - Crystal structure

KW - Enzyme catalysis

KW - Enzyme mechanism

KW - Ferulic acid esterase

KW - Molecular docking

KW - Molecular dynamics

KW - Structure-function

U2 - 10.1074/jbc.RA119.009523

DO - 10.1074/jbc.RA119.009523

M3 - Journal article

VL - 294

SP - 17339

EP - 17353

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 46

M1 - jbc.RA119.009523

ER -