Domain Architecture Shapes Function and Engineering in a Modular Bacterial Galactose Oxidase

Galactose oxidases (GalOxs) are copper radical oxidases that catalyze the selective oxidation of primary alcohols and constitute attractive platforms for carbohydrate biocatalysis. Although fungal AA5_2 enzymes have been extensively characterized, bacterial representatives remain unexplored, and the contribution of appended carbohydrate-binding modules (CBMs) to redox enzyme function is poorly understood. Here, we report the first biochemical and structural characterization of a bacterial galactose oxidase, Ps GalOx from Pseudarthrobacter siccitolerans , revealing a previously unexplored bacterial architectural solution within the AA5_2 family. While the catalytic Cu(II)–tyrosyl radical center is conserved, Ps GalOx displays a divergent modular architecture comprising two tandem N-terminal CBM32 domains. Functional analyses reveal pronounced asymmetry between these duplicated modules: one enhances galactan binding but compromises solubility, whereas the second primarily contributes to structural stabilization. Removal of the N-terminal CBM markedly increases soluble expression without affecting intrinsic catalytic parameters toward D-galactose, demonstrating that enzyme performance can be tuned independently of the conserved catalytic core. Consistently, adaptive engineering trajectories preferentially target domain-scale rearrangements within the CBM region, indicating that selection initially acts on modular architecture to improve physicochemical properties such as solubility, rather than directly enhancing catalytic turnover. These findings expand the structural landscape of AA5_2 oxidases and establish domain architecture as a primary determinant of solubility, substrate recognition, and catalytic output in modular copper radical oxidases.