Active Site Residues Contribute to the Multistep Dynamics of Dicyclotyrosine Bound to CYP121A1 of <i>Mycobacterium tuberculosis</i>

Cytochrome P450 121A1 (CYP121A1) of <i>Mycobacterium tuberculosis</i> is involved in the essential synthesis of mycocyclosin from its substrate, dicyclotyrosine (cYY). As such, CYP121A1 continues to garner significant interest as a drug target. In this study, all-atom molecular dynamics simulations have been employed to investigate the behavior of bound cYY in wild-type CYP121A1, as well as mutants of the active site aromatic residues F168 and W182 that have previously been characterized in vitro. Of note, simulated changes in cYY orientation align closely with changes in CYP121A1 catalysis described in vitro. For example, the mutant W182Y allows cYY to achieve proximity to the heme, which we posit models a catalytically relevant binding mode. Interestingly, a similar binding mode was observed for a single CYP121 protomer in simulations of the intact dimer. These findings, including in vitro analysis of the active site mutation R386N, inform a model of the multistep dynamics of bound cYY and address multiple unresolved questions raised in prior studies while also informing the development of future drug design by highlighting additional orientations of bound cYY that correlate with CYP121A1 function.