Characterization of the polyene macrolide P450 epoxidase from Streptomyces natalensis that converts de-epoxypimaricin into pimaricin

Abstract

The biosynthesis of the antifungal agent pimaricin by Streptomyces natalensis has been proposed to involve a cytochrome P450 encoded by the gene pimD. Pimaricin is derived from its immediate precursor de-epoxypimaricin by epoxidation of the C-4-C-5 double bond on the macrolactone ring. We have overproduced PimD with a N-terminal His6 affinity tag in Escherichia coli and purified the enzyme for kinetic analysis. The protein showed a reduced CO-difference spectrum with a Soret maximum at 450 nm, indicating that it is a cytochrome P450. Purified PimD was shown to catalyse the in vitro C-4-C-5 epoxidation of 4,5-de-epoxypimaricin to pimaricin. The enzyme was dependent on NADPH for activity with optimal pH at 7.5, and the temperature optimum was 30 degrees C. The kcat value for the epoxidation of de-epoxypimaricin was similar to the values reported for other macrolide oxidases. Enzyme activity was inhibited at high substrate concentration. This is the first time that a polyene macrolide P450 mono-oxygenase has been expressed heterologously and studied. The unique specificity of this epoxidase should be useful for the oxidative modification of novel polyene macrolide antibiotics.

Figure 1. Purification of PimD in E. coli BL21

(A) SDS/PAGE (12% gels) of crude E. coli lysate expressing PimD (lane 1) and after purification by Ni²⁺ chromatography (lane 2). (B) Immunoblot detection of His₆–PimD with anti-His-tag antibodies.

Figure 2. Identification of PimD as an authentic cytochrome P450

(A) Comparison of O₂-binding pockets and haem-binding sequences from some macrolide P450 mono-oxygenases. The asterisk on the left alignment shows the conserved threonine/serine (alanine in EryF) residues that are believed to be involved in O₂ scission, whereas the asterisk on the right indicates the haem-binding cysteine residue. Conserved amino acids are shown in bold and are highlighted. Numbers indicate amino acid residues from the N-terminus of the protein. AmphL, St. nodosus P450 involved in amphotericin biosynthesis (GenBank® accession number AAK73504); NysL, St. noursei P450 involved in nystatin biosynthesis (GenBank® accession number AAF71769); EryF (GenBank® accession number Q00441) and EryK (GenBank® accession number P48635), P450s from Sa. erythraea involved in erythromycin biosynthesis; PicK, methymycin/picromycin hydroxylase from St. venezuelae (GenBank® accession number AAC64105). (B) CO difference spectrum of purified PimD. The continuous line indicates the absorbance spectrum of the pure protein in storage buffer. The dotted line denotes the spectrum of the protein reduced with Na₂S₂O₄. The reduced CO-difference spectrum is indicated by a broken line.

Figure 3. In vitro conversion of de-epoxypimaricin into pimaricin by PimD

The results of analytical HPLC after reaction with different enzyme concentrations are shown as follows: (A), no enzyme; (B) 25 nM PimD; (C) 50 nM PimD; (D) 100 nM PimD. Reactions were carried out for 30 min. Chromatographic peaks corresponding to 4,5-de-epoxypimaricin and pimaricin are indicated by arrows.

Figure 4. Least-squares fit of initial rate data

Initial rates were determined by applying a quadratic fit to the linear region of the time trace. The data were then fitted to the equation v=V_max/{1+(K_m/[S])+([S]/K_i)}.