Mycobacteria use specialized ESX secretion systems to transport proteins across their cell membranes in order to manipulate their environment. In pathogenic Mycobacterium tuberculosis there are five paralogous ESX secretion systems, named ESX-1 through ESX-5. Each system includes a subtilisin-like protease (mycosin or MycP) as a core component essential for secretion. Here we report crystal structures of MycP(1) and MycP(3), the mycosins expressed by the ESX-1 and ESX-3 systems, respectively. In both mycosins the putative propeptide wraps around the catalytic domain and does not occlude the active site. The extensive contacts between the putative propeptide and catalytic domain, which include a disulfide bond, suggest that the N-terminal extension is an integral part of the active mycosin. The catalytic residues of MycP(1) and MycP(3) are located in a deep active site groove in contrast with an exposed active site in majority of subtilisins. We show that MycP(1) specifically cleaves ESX-1 secretion-associated protein B (EspB) in vitro at residues Ala358 and Ala386. We also systematically characterize the specificity of MycP(1) using peptide libraries, and show that it has evolved a narrow specificity relative to other subtilisins. Finally, comparison of the MycP(1) and MycP(3) structures suggest that both enzymes have stringent and different specificity profiles that result from the structurally distinct active site pockets, which could explain the system specific functioning of these proteases. (C) 2013 Elsevier Inc. All rights reserved.