Mycobacterium tuberculosis (M. tb.) and Mycobacterium ulcerans (M. ulc.) are pathogenic bacteria that are the cause of tuberculosis (TB) and Buruli Ulcer respectively, two globally distributed diseases[1],[2]. An increase in the number of cases and proportion of strains of multi drug resistant TB (MDR-TB) has become an urgent public health concern[1]. A significant body of literature has demonstrated that the M. tb. cholesterol catabolism pathway gene cluster igr is essential for chronic infection[3] . The P450 enzyme CYP125A1 from M. tb. is a key component of this essential gene cluster whose function is to initiate cholesterol side chain degradation[4] . There is another P450 enzyme in the M. tb. genome, CYP142A1 which is also able to oxidise cholesterol and provide back-up functionality to CYP125A1, albeit with less efficiency[5]. There are homologs of these P450s in M. ulc, namely CYP125A7 and CYP142A3. An understanding of how these enzymes metabolise cholesterol and the development of rationally designed inhibitors of their function represents a novel strategy for anti-mycobacterial drug development. Furthermore, a comparison of the substrate selectivity, catalytic efficiency and structure of the CYP125 and CYP142 enzymes between mycobacterial species can potentially shed light on the evolutionary history of the differing pathogenicity between M. tb. and M. ulc.