Characterization of the Orphan Cytochrome P450 CYP135B1 from <i>Mycobacterium tuberculosis</i>: Involvement in Metabolism but Not in the Antibacterial Activity of the Antitubercular Drug SQ109

The rise of multidrug-resistant tuberculosis (TB) has increased the need for new antitubercular (anti-TB) drugs and the identification of novel drug targets. One promising target is Mycobacterium tuberculosis (Mtb) cytochrome P450 enzymes (P450s). This study focuses on the characterization of CYP135B1, a prevalent Mtb P450. Using a combination of microbiology, genomics, bioinformatics, docking, spectroscopy, and mass spectrometry, researchers successfully expressed, purified, and characterized CYP135B1. A 3D model was built with AlphaFold 3. The enzyme displayed typical features of P450 proteins and showed strong binding to imidazole derivatives. Notably, CYP135B1 metabolized the anti-TB drug SQ109 by inserting oxygen into its geranyl moiety in a manner distinct from CYP124A1. However, genetic studies using a ΔCYP135B1 mutant strain revealed that CYP135B1 is not required for SQ109’s antibacterial activity, as its deletion did not affect drug efficacy despite CYP135B1 metabolizes SQ109.