Engineering a Modular Bacterial Galactose Oxidase through CBM Recombination and Second-Shell Substitutions

Galactose oxidases (GalOxs) are copper-radical enzymes that selectively oxidize primary alcohols, yet the structure–function determinants governing their catalytic efficiency and substrate scope remain incompletely understood, particularly in bacterial enzymes. Here, we combine biochemical and structural characterization with directed evolution of PsGalOx from Pseudarthrobacter siccitolerans. Spectroscopic analysis showed that PsGalOx can form the catalytically competent Cu(II)–tyrosyl radical state. The enzyme also displays an unusual modular architecture comprising two tandem carbohydrate-binding modules (CBMs). The enzyme preferentially oxidizes d-galactose and d-galactose-containing oligo- and polysaccharides. Functional analyses reveal distinct roles for the two CBM32 domains: CBM32-I enhances polysaccharide binding but limits soluble expression, whereas CBM32-II primarily contributes to protein stability. During directed evolution with d-galactose, recombination between the homologous CBMs generated chimeric single-CBM variants enriched primarily through increased soluble production rather than intrinsic catalytic improvement. Subsequent screening with benzyl alcohol as a model noncarbohydrate substrate selected second-shell substitutions near the copper center that increased intrinsic catalytic turnover and broadened substrate scope. The final variant, 14G3, exhibited approximately 40-, 900-, and 1800-fold higher catalytic efficiencies (kcat/Km) than WT toward d-galactose, 5-hydroxymethylfurfural, and benzyl alcohol, respectively, while retaining the primary copper coordination geometry. These findings identify accessory-domain architecture and second-shell composition as distinct engineering levers for improving soluble enzyme production and catalytic performance in a modular bacterial copper radical oxidase (CRO).