Journal of Biological Chemistry
Volume 289, Issue 46, 14 November 2014, Pages 32186-32200
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Protein Structure and Folding
Molecular Mechanisms Associated with Xylan Degradation by Xanthomonas Plant Pathogens*

https://doi.org/10.1074/jbc.M114.605105Get rights and content
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Xanthomonas pathogens attack a variety of economically relevant plants, and their xylan CUT system (carbohydrate utilization with TonB-dependent outer membrane transporter system) contains two major xylanase-related genes, xynA and xynB, which influence biofilm formation and virulence by molecular mechanisms that are still elusive. Herein, we demonstrated that XynA is a rare reducing end xylose-releasing exo-oligoxylanase and not an endo-β-1,4-xylanase as predicted. Structural analysis revealed that an insertion in the β7-α7 loop induces dimerization and promotes a physical barrier at the +2 subsite conferring this unique mode of action within the GH10 family. A single mutation that impaired dimerization became XynA active against xylan, and high endolytic activity was achieved when this loop was tailored to match a canonical sequence of endo-β-1,4-xylanases, supporting our mechanistic model. On the other hand, the divergent XynB proved to be a classical endo-β-1,4-xylanase, despite the low sequence similarity to characterized GH10 xylanases. Interestingly, this enzyme contains a calcium ion bound nearby to the glycone-binding region, which is required for catalytic activity and structural stability. These results shed light on the molecular basis for xylan degradation by Xanthomonas and suggest how these enzymes synergistically assist infection and pathogenesis. Our findings indicate that XynB contributes to breach the plant cell wall barrier, providing nutrients and facilitating the translocation of effector molecules, whereas the exo-oligoxylanase XynA possibly participates in the suppression of oligosaccharide-induced immune responses.

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*

This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo Grants 10/51890-8, 13/13309-0, and 14/07135-1; Conselho Nacional de Desenvolvimento Científico e Tecnológico Grants 476043/2011-5 and 308092/2012-0; and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior.

The atomic coordinates and structure factors (codes 4PMU, 4PMV, 4PMX, 4PMY, 4PMZ, and 4PN2) have been deposited in the Protein Data Bank (http://wwpdb.org/).

1

These authors contributed equally to this work.

3

The abbreviations used are:

    CWDE

    cell wall-degrading enzyme

    T2SS

    type II secretion system

    T3SS

    type III secretion system

    Xac

    Xanthomonas axonopodis pv. citri

    Xcc

    X. campestris pv. campestris

    Xoo

    X. orizae pv. orizae

    Xcv

    X. campestris pv. vesicatoria

    CE-LIF

    capillary electrophoresis coupled to laser-induced fluorescence

    RMSD

    root mean square deviation

    SAXS

    small angle x-ray scattering.