Takamatsu et al. used a bioinformatics approach to identify orthologous genes encoding epi-isozizaene synthase and CYP170 in nine bacterial strains, and indicated that all but one is translationally coupled to a downstream CYP gene . Each putative epi-isozizaene synthase was identified by the high degree of protein similarity between all proteins and the retention of canonical motifs essential for epi-isozizaene synthase activity identified in the resolved crystal structure of the S. coelicolor A3(2) enzyme. Concurrently, we undertook a bioinformatics search to determine the presence of other bifunctional CYPs in the databases. Hence, a blast search with the protein sequence of the S. coelicolor A3(2) CYP, CYP170A1, as a query identified 10 CYPs with homology to CYP170A1 in the following streptomycete genomes: S. lividans, S. viridochromogenes, S. sviceus, S. avermitilis, S. ghanaensis, S. griseoflavus, Streptomyces sp. E14, Streptomyces sp. SPB74, Streptomyces sp. SPB78, and S. albus. All CYPs identified contained the signature CYP motifs used for identification: the heme-binding domain FXXGXRXCXX and the EXXR motif involved in stabilization of heme during incorporation and CYP folding. Amino acid sequence alignment of seven CYP170 family members revealed a high degree of identity with CYP170A1 (S. lividans, 99%; S. viridochromogenes, 86%; S. ghanaensis, 81%; S. sviceus, 80%; S. avermitilis, 80%; S. griseoflavus, 75%; and Streptomyces sp. E14, 74%). Additionally, protein sequence alignment revealed conservation of the terpene synthase active site moonlighting on each of the seven CYPs in an analogous fashion to CYP170A1. All seven CYPs contain the conserved Mg2+-binding motifs [DDXXTXXXE250(242–250) and DDXXD(253–257) in CYP170A1)], which form part of a four-helical barrel responsible for terpene synthase activity, suggesting that all of these CYPs are bifunctional. The only variation found in the Mg2+-binding motifs of six of the seven CYPs is in the ortholog from S. sviceus, which contains the changes DDXXTXXXA(237–245) and DAXXD(248–252), but these are not predicted to alter Mg2+ binding, owing to the conservative nature of both substitutions. The least related CYP170 orthologs coupled to the epi-isozizaene synthase gene were found in Streptomyces sp. SPB74, Streptomyces sp. SPB78, and S. albus, each showing approximately 56% identity with CYP170A1. Within these three CYP170 orthologs, the Mg2+-binding motifs, which form the moonlighting terpene synthase site in CYP170A1, show differences from those in the other CYP170A orthologs, and these CYPs represent a new CYP170 subfamily – CYP170B. In the S. albus CYP170 (CYP170B1) sequence, the Mg2+ motifs are DDXXDXXXT(236–244) and DGXXR(247–251), and there is reduced similarity in the α-helical barrel, suggesting that CYP170B1 may not be bifunctional. Similarly, in Streptomyces sp. SPB74 and Streptomyces sp. SPB78 CYP170Bs, the Mg2+ motifs are DDXXSXXXH(240–248) in both, and GEXXE(251–255) in Streptomyces sp. SPB74 and GEXXE(251–255) in Streptomyces sp. SBP78, again suggesting a loss of bifunctionality.
Furthermore, examination of the genes surrounding the albaflavenone operon in each streptomycete reveals a high degree of conservation. Directly upstream, the sigR and anti-sigR genes encode sigma factors that coordinate expression of the disulfide stress regulon. Additional genes that are highly conserved in each species include a gene that encodes an integral membrane protein that may play a role in albaflavenone export and a gene that encodes a tetratricopeptide repeat protein (TRP); TRPs constitute a family of proteins that have been shown to form scaffolds to mediate protein–protein interactions . TRP may be involved in forming a complex between epi-isozizaene synthase and CYP170, allowing efficient production of albaflavenone through the channeling of epi-isozizaene between both enzymes.