*2.3. CYP139 Family Has Unique Amino Acid Patterns at CXG Motif*

In a study by Syed and Mashele [28], analysis of the P450 signature motifs, EXXR and CXG, among different P450 families led to the discovery of amino acid patterns characteristic of a P450 family. The authors proposed that "during the divergence of P450 families from a common ancestor, these amino acids patterns evolved and are retained in each P450 family as a signature of that family" [28]. However, in that study, the CYP139 P450 family is not included. Furthermore, identification of a large number of CYP139A P450s, in this study, gives us an opportunity to identify CYP139 P450 family characteristic amino acid patterns at EXXR and CXG motifs, if any.

Analysis of EXXR and CXG motifs in 894 CYP139A P450s revealed that the CYP139 P450 family EXXR domain is absolutely conserved with amino acid patterns E-T-L-R, whereas, eight amino acids are invariantly conserved in CXG motifs with amino acid patterns of F-S-G(96%)/A(4%)-G-L-H-R-C-I(96%)/V(4%)-G (Figure 3). It is interesting to note that the CYP139 P450 family EXXR motif amino acid pattern absolutely matched with the CYP5 family [28] and amino acid patterns at the CXG motif were unique and not matched with any P450 families described in the literature [25,28,29]. The CYP139 P450 family amino acid patterns at the EXXR and CXG motifs further strongly support the above hypothesis proposed by Syed and Mashele [28].

**Figure 3.** Analysis of amino acid patterns at the EXXR and CXG motif in CYP139 P450 family. In total 894 CYP139 P450 sequences were analysed for EXXR and CXG signature sequences.
