*2.5. CYP139A P450s Involved in the Synthesis of Secondary Metabolites in Mycobacterial Species*

Comprehensive comparative analysis of CYP139A P450s secondary BGCs in mycobacterial species revealed that CYP139A P450s are indeed involved in the synthesis of different secondary metabolites, as 92% of CYP139A P450s were found to be part of secondary metabolite BGCs (Figures 4 and 5 and Table S2). To understand the role of CYP139A P450s in mycobacterial species' physiology well, a functional comparison of CYP139A P450s gene clusters' homolog secondary metabolites was carried out (Table 2). As shown in Table 2, it is clear that CYP139A P450s are involved in the production of chemicals that have antibacterial, antifungal, antiviral and antitumor properties. Interestingly, some of these metabolites in fact showed antimycobacterial activity (Table 2). This indicates that CYP139A P450s are possibly helping mycobacterial species to kill other bacteria, including other mycobacterial species, thus gaining the upper hand in the niche area for their survival. It is interesting to note that CYP139A P450s are present only in MTBC, NTM and MAC categories, but not present in SAP, MCAC or MCL. This necessitates understanding its role in mycobacterial species when they are surviving in hosts such as humans or other animals. In this direction, analysis of some secondary metabolite functions pointed out that some secondary metabolites are certainly helping mycobacterial species to survive in their hosts. For example, MAR/MAP BGC products are found to be part of the cell envelope in *M. marinum*, possibly complicating its access to host immune system or drug actions [30]; Akaeolide has cytotoxic activity against fibroblasts, suggesting it may play a role in tissue weakening in the host [31]; JBIR-100 exhibits cytotoxic activities and inhibition of proton pumps such as vacuolar-type ATPases (V-ATPases) activities and is thus linked with an increasing number of diseases such as osteopetrosis, male infertility and renal acidosis [32,33]. Lorneic acid A inhibits phosphodiesterase PDE5 blocking the degradation of cGMP [34] and thus it might be playing a role in pulmonary hypertension. Meridamycin has been found to bind FK506-binding proteins (FKBP12) [35]. FKBP12 proteins play a key role in

regulating fundamental aspects of cell biology and have been found to be critical in mice survival [36]. Nigericin inhibits the Golgi functions in eukaryotic cells and is a well-known activator of the NLRP3 inflammasome [37–39], indicating bacterial infection. One secondary metabolite, namely mycolactone, a lipid-like toxin with cytotoxic, immunosuppressive and tissue necrosis activity, has been shown to be involved in the development of Buruli ulcer by *M. ulcerans* [40].


#### **Table 2.** Functional analysis of homolog CYP139A P450 gene clusters.

### **3. Materials and Methods**

#### *3.1. Mycobacterial Species and Genome Databases*

In total, 1111 mycobacterial species genomes that are available for public use (as of 12 June 2018) at Integrated Microbial Genomes & Microbiomes (IMG/M) [68] were used in the study (Table S1). Mycobacterial species used in the study, along with their name, genome ID and individual genome database links, were presented in Table S1.

#### *3.2. Genome Data Mining and Annotation of CYP139 P450s*

The *M. tuberculosis* H37Rv CYP139A1 (Rv1666c) P450 sequence has been blasted with the default settings against individual mycobacterial species genomes at IMG/M [68]. However, each time, only 20 mycobacterial species were selected for BLAST analysis. The hit proteins with more than 40% identity were selected and then subjected to BLAST analysis at the P450 BLAST server (https://ksyed.weebly.com/p450-blast.html) to identify the homolog P450. Hit proteins were then grouped into families and subfamilies based on the International Cytochrome P450 Nomenclature criteria, i.e., P450s showing >40% identity were assigned to the same P450 family and P450s that showed >55% identity were grouped under the same P450 subfamily [69–71]. Protein with more than 90% identity considered as ortholog and assigned the same subfamily number.

#### *3.3. Phylogenetic Analysis of CYP139A P450s*

The phylogenetic tree of CYP139 family members was built with *M. tuberculosis* CYP51B1 (Rv0764c) protein as outgroup. First, the protein sequences were aligned by MAFFT v6.864 [72], embedded on the Trex web serve [73]. Then, the alignments were automatically subjected to infer the best tree by the Trex web server with its embedded weighting procedure. Finally, the tree was visualised and colored by iTOL (http://itol.embl.de/about.cgi) [74].

#### *3.4. Analysis of Homology and Amino Acid Conservation*

Analysis of percentage identity among CYP139A P450s from species belonging to MAC and NTM categories was carried out as described elsewhere [23,29]. Briefly, the percentage identity between CYP139 P450s was determined using the Clustal Omega [75]. The Clustal Omega percentage identity matrix was downloaded and pasted into an Excel sheet by converting the text into a column option.

Amino acid conservation among CYP139A P450s was carried out following the method described elsewhere [23,25,29]. Briefly, CYP139 P450s were subjected to PROMALS3D [26] to identify invariantly conserved amino acids [27]. The conservation index follows numbers from 5–9, where 9 is the invariantly conserved amino acid across the sequences. The total number of conserved residues indicated by number 9 was recorded. The conserved nature of the CYP139 family was compared to other P450 families from different biological kingdoms, as reported elsewhere [23,25].
