The study of the genus Streptomyces is of particular interest because it produces a wide array of clinically important bioactive molecules. The genomic sequencing of many Streptomyces species has revealed unusually large numbers of cytochrome P450 genes, which are involved in the biosynthesis of secondary metabolites. Many macrolide biosynthetic pathways are catalyzed by a series of enzymes in gene clusters including polyketide and non-ribosomal peptide synthesis. In general, Streptomyces P450 enzymes accelerate the final, post-polyketide synthesis steps to enhance the structural architecture of macrolide chemistry. In this review, we discuss the major Streptomyces P450 enzymes research focused on the biosynthetic processing of macrolide therapeutic agents, with an emphasis on their biochemical mechanisms and structural insights.

It has been recognized for >50 years that cytochrome b₅ (b₅) stimulates some cytochrome P450 (P450)-catalyzed oxidations, but the basis of this function is still not understood well. The strongest stimulation of catalytic activity by b₅ is in the P450 17A1 lyase reaction, an essential step in androgen synthesis from 21-carbon (C21) steroids, making this an excellent model system to interrogate b₅ function. One of the issues in studying b₅-P450 interactions has been the limited solution assay methods. We constructed a fluorescently-labeled variant of human b₅ that can be used in titrations. The labeled b₅ bound to wild-type P450 17A1 with a K_d of 2.5 nM and rapid kinetics, on the order of 1 s^-1. Only weak binding was observed with the clinical P450 17A1 variants E305G, R347H, and R358Q; these mutants are deficient in lyase activity, which has been hypothesized to be due to attenuated b₅ binding. K_d values were not affected by the presence of P450 17A1 substrates. A peptide containing the P450 17A1 Arg-347/Arg-358 region attenuated Alexa 488-T70C-b₅ fluorescence at higher concentrations. The addition of NADPH-P450 reductase (POR) to an Alexa 488-T70C-b₅:P450 17A1 complex resulted in a concentration-dependent, partial restoration of b₅ fluorescence, indicative of a ternary P450:b₅:POR complex, which was also supported by gel filtration experiments. Overall, these results are interpreted in the context of a dynamic and tight P450 17A1:b₅ complex that also binds POR to form a catalytically competent ternary complex, and variants that disrupt this interaction have low catalytic activity.

Cytochrome P450 2A6 (CYP2A6) catalyzes important metabolic reactions of many xenobiotic compounds, including coumarin, nicotine, cotinine, and clinical drugs. Genetic polymorphisms of CYP2A6 can influence its metabolic activities. This study analyzed the functional activities of six CYP2A6 allelic variants (CYP2A6*5, *7, *8, *18, *19, and *35) containing nonsynonymous single-nucleotide polymorphisms. Recombinant variant enzymes of CYP2A6*7, *8, *18, *19, and *35 were successfully expressed in Escherichia coli and purified. However, a P450 holoenzyme spectrum was not detected for the CYP2A6*5 allelic variant (G479V). Structural analysis shows that the G479V mutation may alter the interaction between the A helix and the F—G helices. Enzyme kinetic analyses indicated that the effects of mutations in CYP2A6 allelic variants on drug metabolism are dependent on the substrates. In the case of coumarin 7-hydroxylation, CYP2A6*8 and *35 displayed increased Km values whereas CYP2A6*18 and *19 showed decreased kcat values, which resulted in lower catalytic efficiencies (kcat/Km). In the case of nicotine 5-oxidation, the CYP2A6*19 variant exhibited an increased Km value, whereas CYP2A6*18 and *35 showed much greater decreases in kcat values. These results suggest that individuals carrying these allelic variants are likely to have different metabolisms for different CYP2A6 substrates. Functional characterization of these allelic variants of CYP2A6 can help determine the importance of CYP2A6 polymorphisms in the metabolism of many clinical drugs.