The structure and peroxidase activity of a 33‐kDa catalase‐related protein from Mycobacterium avium ssp. paratuberculosis

True catalases are tyrosine‐liganded, usually tetrameric, hemoproteins with subunit sizes of ∼55–84 kDa. Recently characterized hemoproteins with a catalase‐related structure, yet lacking in catalatic activity, include the 40–43 kDa allene oxide synthases of marine invertebrates and cyanobacteria. Herein, we describe the 1.8 Å X‐ray crystal structure of a 33 kDa subunit hemoprotein from Mycobacterium avium ssp. paratuberculosis (annotated as MAP‐2744c) , that retains the core elements of the catalase fold and exhibits an organic peroxide‐dependent peroxidase activity. MAP‐2744c exhibits negligible catalatic activity, weak peroxidatic activity using hydrogen peroxide (20/s) and strong peroxidase activity (∼300/s) using organic hydroperoxides as co‐substrate. Key amino acid differences significantly impact prosthetic group conformation and placement and confer a distinct activity to this prototypical member of a group of conserved bacterial “minicatalases”. Its structural features and the result of the enzyme assays support a role for MAP‐2744c and its close homologues in mitigating challenge by a variety of reactive oxygen species.