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Mycobacteria are important causes of infectious diseases. Although Mycobacterium tuberculosis and Mycobacterium leprae, two slow-growing species, are responsible for the most serious diseases, some fast-growing species, such as Mycobacterium avium, Mycobacterium kansasii, Mycobacterium chelonae, and Mycobacterium fortuitum, may cause opportunistic infections among AIDS patients (24). In addition, M. fortuitum has been reported to be responsible for a wide spectrum of clinical diseases, such as skin or soft tissue infections following surgery, pulmonary infections, accidental penetrating trauma, and wounds that come in contact with soil or water contaminated with these mycobacteria, though nosocomial infections are by far the most common (41, 44). The cell wall of mycobacteria contains mycolic acid, arabinogalactan, and peptidoglycan, forming a covalent complex. The influx of small hydrophilic agents through the resulting low-permeability envelope is extremely slow and is thought to be one of the major factors involved in the resistance of mycobacteria to β-lactam antibiotics (21, 41). β-Lactamase production, catalyzing β-lactam antibiotic hydrolysis, appears to be the second mechanism by which mycobacteria express β-lactam resistance (9, 21, 35). Chromosomally encoded β-lactamases have been detected in most, but not all, mycobacterial species, including M. fortuitum and M. tuberculosis (43). The class A β-lactamase of M. fortuitum (MFO) is a chromosomally encoded broad-spectrum β-lactamase hydrolyzing both cephalosporins and penicillins. Its substrate profile includes ureidopenicillins (piperacillin, azlocillin, and mezlocillin), carbenicillin, cephalothin, cefotaxime, and cefuroxime, but not ceftazidime. Cefoxitin, dicloxacillin, imipenem, and aztreonam are poorly recognized by the enzyme, and the protein is inhibited by the penem inhibitor BRL42715 and, to a lesser extent, clavulanic acid. The specificity profile is similar overall to those of CTX-M-type enzymes; some TEM-derived extended-spectrum β-lactamases (ESBLs); the chromosomally encoded ESBLs from Citrobacter sedlakii and Yersinia enterocolitica; and β-lactamases identified in the genera Nocardia, Kluyvera, and Burkholderia (1, 12, 31, 33, 35, 37). At the amino-acid level, the mature protein shares 70% identity with the β-lactamase of Mycobacterium smegmatis and between 40 and 45% with the aforementioned proteins. MFO also shares 41% amino acid identity with the ESBL Toho-1. Many recent studies have been concerned with the structure solution of TEM, SHV, or CTX-M-type ESBLs, but few structural data are available on chromosomally encoded broad-spectrum class A β-lactamases. MFO is the first solved structure of a mycobacterial β-lactamase. Its structure-activity relationship was analyzed on the basis of a comparison between its structure and recently solved ESBL structures (5, 27, 29, 38).