Functional analysis of MycCI and MycG, cytochrome P450 enzymes involved in biosynthesis of mycinamicin macrolide antibiotics

Abstract

Macrolides are a class of valuable antibiotics that include a macrolactone ring, at least one appended sugar unit, and, in most cases, additional hydroxyl or epoxide groups installed by cytochrome P450 enzymes. These functional groups contribute to structural diversification and serve to improve the bioactivity profiles of natural products. Here, we have characterized in vitro two P450 enzymes from the mycinamicin biosynthetic pathway of Micromonospora griseorubida. First, MycCI was characterized as the C21 methyl hydroxylase of mycinamicin VIII, the earliest macrolide form in the postpolyketide synthase tailoring pathway. Moreover, we established that optimal activity of MycCI depends on the native ferredoxin MycCII. Second, MycG P450 catalyzes consecutive hydroxylation and epoxidation reactions with mycinamicin IV as initial substrate. These reactions require prior dimethylation of 6-deoxyallose to mycinose for effective conversion by the dual function MycG enzyme.

Figure 1. Mycinamicin post-PKS biosynthetic pathway and organization of the mycinamicin biosynthetic gene cluster

Methyl groups introduced by O-methyltransferases, and oxidation steps catalyzed by cytochrome P450 enzymes are highlighted in green and red, respectively. Bold arrows represent the main pathway, whereas thin arrows indicate a shunt pathway. The dashed arrow indicates low-level conversion as previously reported (Inouye et al., 1994). Color codes in the myc gene cluster are: Red, cytochrome P450 genes; purple, ferredoxin gene; indigo blue, polyketide synthetase genes; blue, glycosyltransferase genes; orange, O-methyltransferase genes; green, deoxysugar biosynthetic genes; yellow, self-resistance gene (rRNA methyltransferase gene).

Figure 2. Phylogenetic tree of macrolide biosynthetic P450 monooxygenases

The selected cytochrome P450s include OleP (accession # AAA92553) [oleandomycin pathway], ChmHI and ChmPI (accession # AAS79447 and AAS79453) [chalcomycin pathway], MycCI and MycG [mycinamicin pathway] (accession # BAC57023 and BAA03672), AmphL and AmphN (accession # AAK73504 and AAK73509) [amphotericin pathway], NysL and NysN (accession # AAF71769 and AAF71771) [nystatin pathway], PimD and PimG (accession # CAC20932 and CAC20928) [pimaricin pathway], TylHI and TylI (accession # AAD41818 and AAA21341) [tylosin pathway], EryF and EryK (accession # AAA26496 and YP-001102980) [erythromycin pathway], and PikC (accession # AAC68886) [pikromycin pathway]. The numbers in parentheses indicate the macrolactone ring size of the corresponding P450 substrate. Unless otherwise specified, all selected P450 enzymes are hydroxylases. The epoxidases are noted in italics. The enzymes marked with an asterisk are presumed to mediate carboxylic acid formation. MycCI and MycG are highlighted in bold.

Figure 3. Synthetic scheme for M-VIII

Figure 4. In vitro M-VIII conversions catalyzed by MycCI (all LC traces were analyzed at 280 nm)

(1) negative control, M-VIII + boiled MycCI; (2) M-VIII + MycCI + spinach ferredoxin; (3) M-VIII + MycCI + MycCII-NH; (4) M-VIII + MycCI + MycCII-wt; (5) M-VII standard. The right diagram shows M-VIII conversion ratios calculated by using equation 1-AUC_{unreacted M-VIII}/AUC_{total M-VIII} (AUC: area under curve) based on the corresponding LC-traces in parallel. In addition, dissociation constants (K_d) of the spinach and MycCII ferredoxins toward MycCI are shown.

Figure 5. LC-MS analysis of in vitro conversions catalyzed by MycG (all LC traces were analyzed at 280 nm unless otherwise specified)

(A) MycG reaction using M-IV as substrate: (1) negative control, M-IV + boiled MycG; (2) M-IV + MycG. The right panel shows the mass spectra of peak a and b; (3) M-II standard amplified 20 × due to its poor absorbance at 280 nm; (4) M-V standard; (5) M-I standard amplified 20 ×. (B) MycG reaction using M-V as substrate: (1) negative control, M-V + boiled MycG; (2) M-V + MycG. The inset shows the 240 nm LC trace, under which M-II has better absorbance. The right panel shows the mass spectrum of peak c; (3) M-II standard amplified 20 ×. (C) MycG reactions using M-III and M-VI as alternative substrates: (1) negative control, M-III + boiled MycG; (2) M-III + MycG; (3) negative control, M-VI + boiled MycG; (4) M-VI + MycG. LC traces (3) and (4) amplified 20 × compared to (1) and (2) to visualize the peak e in trace amount. The right panel shows the mass spectra of peak d and e.