Evolvable Carbohydrate-Binding Modules Shape Function and Engineering of a Bacterial Galactose Oxidase

Carbohydrate-binding modules (CBMs) are widespread auxiliary domains in carbohydrate-active enzymes, yet their contribution to enzyme function and evolvability remains unclear, particularly in redox enzymes. Here, we report the first biochemically and structurally characterized bacterial galactose oxidase. Unlike its fungal counterparts, this enzyme is produced in an active form containing a catalytic Cu(II)–tyrosyl radical cofactor and two N-terminal CBM32 domains. Biochemical, structural, and computational analyses reveal functional asymmetry between the duplicated CBMs, which differentially modulate soluble expression, stability and polysaccharide binding. Deletion of the first CBM markedly enhances solubility while preserving catalytic competence, and binding assays identify this module as the dominant contributor to galactan affinity. Directed evolution promotes CBM truncation and recombination, yielding functional chimeric variants and uncovering a residue influencing polysaccharide recognition. These findings expand the diversity of copper radical oxidases and highlight domain architecture as an evolvable determinant of redox enzyme behavior.