Shifting the substrate scope of dimeric pyranose oxidase from monosaccharide to glycoside preference through oligomeric state modification

TL;DR

Researchers modified a dimeric pyranose oxidase to prefer glycosides over monosaccharides by altering its oligomeric state.

Contribution

The study demonstrates that modifying the oligomeric state of pyranose oxidase can shift its substrate preference from monosaccharides to glycosides.

Findings

Deleting domains in KaPOx led to monomeric variants that preferred glycosides like phlorizin over monosaccharides.

Monomeric KaPOx variants showed 24 × 10⁶ higher catalytic efficiency for phlorizin compared to d-xylose.

Dimeric KaPOx had no activity towards glycosides due to steric hindrance and active site inaccessibility.

Abstract

Pyranose oxidase (POx) and C‐glycoside oxidase (CGOx) are FAD‐dependent oxidoreductases belonging to the glucose‐methanol‐choline oxidoreductase superfamily and share the same sequence space. Despite a shared structural fold, these two members possess homologous domains that enable (arm and head domain) or disable (insertion‐1 domain and barrel‐shaped bottom) oligomerization. POxs with a higher oligomerization state (dimeric or tetrameric) exclusively catalyze the oxidation of monosaccharides (d‐glucose, d‐xylose). In contrast, the monomeric state of POxs/CGOxs is observed to prefer glycosides (homoorientin, phlorizin) and has low activity with free monosaccharides. We aimed to engineer dimeric POx from Kitasatospora aureofaciens (KaPOx) to form a functional monomer, and monomeric POx/CGOx from Streptomyces canus (ScPOx) to a dimeric structure. Deletion of the head and arm domains of the KaPOx subunit resulted in enzyme variants with a less hydrophobic surface, thus affecting its oligomerization. These monomeric KaPOx variants KaPOx_xal and KaPOx_xalh resembled monomeric wild‐type POxs/CGOxs and preferred glycosides as substrates over monosaccharides with catalytic efficiencies for phlorizin being 24 × 106 higher compared to those for d‐xylose. The wild‐type dimeric KaPOx showed no activity towards glycosides. We hypothesize that KaPOx_xalh is unable to react with monosaccharides because the introduced mutations alter the positions of monosaccharide‐binding residues. The inability of KaPOx to react with glycosides is likely caused by steric hindrance and the inaccessibility of the active site to bulky glycosides due to dimerization. The attempt to engineer ScPOx into a dimeric structure failed at the stage of soluble expression, likely due to exposed hydrophobic patches and aggregation.

Pyranose oxidase (POx) and C‐glycoside oxidase (CGOx) are FAD‐dependent oxidoreductases that share the same sequence space. POxs with a higher oligomerization state exclusively catalyze the oxidation of monosaccharides, whereas monomeric POxs/CGOxs show preference towards glycosides. We engineered the dimeric POx from Kitasatospora aureofaciens (KaPOx) to form a monomer. Deletion of the oligomerization domains of KaPOx resulted in monomeric variants, which resemble bacterial wild‐type POxs/CGOxs and prefer glycosides as substrates.

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