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<p>Xylanases are central to lignocellulosic biomass degradation, yet current methods lack the specificity to resolve how enzymes distinguish complex xylan structures decorated with arabinofuranose (Ara<i>f</i>) and 4-<i>O</i>-methyl-glucuronic acid (MeGlcA). Here, we report a suite of chemically-defined activity-based probes (ABPs) that enable the selective detection of arabinoxylan- and glucuronoxylan-specific xylanases (AXXs and GXXs). These cyclophellitol-derived ABPs covalently label retaining xylanases at their active sites, allowing precise mapping of substrate specificity across diverse glycoside hydrolase families. Crystallographic and mass spectrometric analyses reveal the molecular basis of probe selectivity, while in-gel and pull-down assays demonstrate their effectiveness in profiling xylanase activities in complex bacterial and fungal proteomes, including cellulosomes. By integrating activity-based protein profiling (ABPP) with sequence similarity networks (SSNs), we further show that xylanase specificity can be predicted from sequence alone, enabling rapid functional annotation of uncharacterized xylanases. This chemoproteomic strategy provides a powerful platform for discovering and engineering substrate-specific enzymes for biomass valorisation, microbial ecology, and biotechnological applications.</p>

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A chemoproteomic biotechnological toolkit for resolving xylanase specificity in decorated xylan

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年6月22日

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  • Thamy L. R. Corrêa,
  • Zirui Li,
  • Olga Moroz,
  • Isabelle B. Pickles,
  • Andrey A. Lebedev,
  • Saeed Akkad,
  • Lianne I. Willems,
  • Jeroen D. C. Codée,
  • Herman S. Overkleeft,
  • Gideon J. Davies

摘要

Xylanases are central to lignocellulosic biomass degradation, yet current methods lack the specificity to resolve how enzymes distinguish complex xylan structures decorated with arabinofuranose (Araf) and 4-O-methyl-glucuronic acid (MeGlcA). Here, we report a suite of chemically-defined activity-based probes (ABPs) that enable the selective detection of arabinoxylan- and glucuronoxylan-specific xylanases (AXXs and GXXs). These cyclophellitol-derived ABPs covalently label retaining xylanases at their active sites, allowing precise mapping of substrate specificity across diverse glycoside hydrolase families. Crystallographic and mass spectrometric analyses reveal the molecular basis of probe selectivity, while in-gel and pull-down assays demonstrate their effectiveness in profiling xylanase activities in complex bacterial and fungal proteomes, including cellulosomes. By integrating activity-based protein profiling (ABPP) with sequence similarity networks (SSNs), we further show that xylanase specificity can be predicted from sequence alone, enabling rapid functional annotation of uncharacterized xylanases. This chemoproteomic strategy provides a powerful platform for discovering and engineering substrate-specific enzymes for biomass valorisation, microbial ecology, and biotechnological applications.