Beyond monofunctionality: structural perspectives on phenol oxidase side activity in a catalase

Catalases are ubiquitous enzymes that protect cells from oxidative stress through the disproportionation of hydrogen peroxide. Although oxidase side activities have been reported for a limited number of catalases, their structural basis and physiological relevance remain poorly understood. This study investigates the catalytic promiscuity of ClCPO, an extracellular fungal catalase from the marine fungus Cladosporium sp., previously identified as a potential degrader of persistent organic pollutants. Recombinant ClCPO was biochemically characterized and shown to catalyse the oxidation of ortho-diphenols to ortho-quinones, bearing hydroxy, ethylamine and single and double chlorine substitutions on the benzene ring, in the absence of hydrogen peroxide, while retaining robust catalase activity. On the contrary, it did not exhibit any cresolase activity in either unsubstituted or chlorinated monophenols, nor phenol oxidative activity on hydroxy-biphenyls. Crystal structures of apo ClCPO and a catechol-bound complex were determined at high resolution, identifying a catechol-binding site located at the junction of the main and secondary access channels to the haem cofactor. Docking simulations suggested an additional candidate ligand-binding region capable of accommodating bulkier compounds, whereas in silico electron/hole-transfer pathway analysis identified recurrent predicted pathway elements connecting the different ligand-binding regions to the haem centre. Structural comparison with previously characterized bifunctional catalases highlights distinct substrate-recognition features and suggests an alternative mechanistic basis for phenol oxidase activity in ClCPO. Together, these findings provide structural insight into the catalytic promiscuity of ClCPO, support a working mechanistic framework for phenol oxidase activity in fungal catalases and expand our understanding of the functional diversity of haem-dependent oxidoreductases.