*3.6. Identification of CYP128 P450 Secondary Metabolite BGCs*

Secondary metabolite BGCs analysis of CYP128 P450s was carried out following the method described elsewhere [31]. Mycobacterial species BGCs listed at the IMG/M website were manually searched for the presence of CYP128 P450s using their gene ID [42]. The BGCs that contained CYP128 P450 were selected and the entire gene cluster sequence was downloaded. The listed BGCs at IMG/M are unspecific and to identify the particular BGC type, the downloaded gene cluster sequence was subjected to secondary metabolite BGC analysis using anti-SMASH [54]. The type of BGC, percentage similarity to a known cluster and the known cluster name were recorded from the anti-SMASH analysis. Standard BGC abbreviation terminology developed by anti-SMASH was used in the study.

#### *3.7. CYP128 Homology Modeling*

The Molecular Operating Environment (Chemical Computing Group) was used to build a 3D model of CYP128A1. The crystallographic structure of the P450 Vitamin D3 hydoxylase bound with vitamin D3 (VD3) (PDB ID: 3VRM) [37] was used as a template, showing 33.8% identity and 48% similarity with the targeted sequence after alignment. Homology modeling of CYP128A1 was performed by setting the number of generated models to 10 and by selecting the final model based on MOE's Generalized Born/Volume Integral (GB/VI) scoring function. During the modeling, the heme group of the template—including the ferric ion—was kept as part of the environment and included in the refinement step. The final model was eventually protonated at neutral pH and minimized using a MOE's built-in protocol.
