Journal of Biological Chemistry
Volume 294, Issue 46, 15 November 2019, Pages 17339-17353
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Enzymology
A carbohydrate-binding family 48 module enables feruloyl esterase action on polymeric arabinoxylanArabinoxylan-binding CBM48

https://doi.org/10.1074/jbc.RA119.009523Get rights and content
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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, wastewater treatment sludge (wts) Fae1A 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.

crystal structure
enzyme catalysis
enzyme mechanism
structure–function
molecular docking
molecular dynamics
arabinoxylan
carbohydrate-binding module
carbohydrate esterase family 1
ferulic acid esterase

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This work was supported by Villum Foundation Grant VKR022796 (to L. L.) and base funding from the Technical University of Denmark. J. B. and K. B. R. M. K. are employees at Novozymes A/S, a company that produces and sells enzymes and microbes for industrial uses.

This article contains Figs. S1–S9 and Table S1.

The protein sequence for the wtsFae1B gene has been deposited in the NCBI Protein Database under NCBI accession no.BK010854.1.

The atomic coordinates and structure factors (codes 6RZO and 6RZN) have been deposited in the Protein Data Bank (http://wwpdb.org/).

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1

Supported by Novo Nordisk Foundation Grant NNF10CC1016517 to the Novo Nordisk Foundation Center for Biosustainability at Technical University of Denmark.

2

Supported by The Independent Research Fund Denmark, Natural Sciences Grant 6108-00476A.

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The abbreviations used are:

    AX

    arabinoxylans

    Araf

    arabinofuranose

    AXOS

    arabinoxylooligosaccharide

    CAZy

    Carbohydrate-Active EnZyme database

    CBM

    carbohydrate-binding module

    CBM48

    carbohydrate-binding module family 48

    CE1

    carbohydrate esterase family 1

    di-FA

    ferulate dehydrodimer

    FA

    ferulic acid

    GH10

    glycoside hydrolase family 10

    r.m.s.d.

    root-mean-square deviation

    SPR

    surface plasmon resonance

    WAX-I

    insoluble wheat arabinoxylan

    Xylp

    xylopyranose

    XA5f2X

    β-d-Xylp-(1→4)-[5-O-feruloyl-α-l-Araf-(1→3)]-β-d-Xylp-(1→4)-β-d-Xylp

    Bis-Tris

    2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol

    Bistris propane

    1,3-bis[tris(hydroxymethyl)methylamino]propane

    RU

    resonance unit

    wts

    wastewater treatment sludge

    DSC

    differential scanning calorimetry.