Biosynthesis of the macrolide antibiotics methymycin and erythromycin

We describe the preparation and intact incorporation of (2S,3R)- (2,3-{dollar}rmsp{lcub}13{rcub}Csb2rbrack{dollar}-2-methyl-3-hydroxypentanoyl N-acetylcysteamine (NAC) thioester, (2S,3R)- (3-{dollar}sp2{dollar}H,3-{dollar}rmsp{lcub}13{rcub}Csb2{dollar}) -2-methyl-3-hydroxypentanoyl NAC thioester, and (4R,5R)- (2,3-{dollar}rmsp{lcub}13{rcub}Csb2rbrack{dollar}-4-methyl-5-hydroxy-2-heptenoyl NAC thioester into the macrolide antibiotics methymycin and neomethymycin. Our results provide further confirmation that the parent macrocyclic lactones of the macrolides are formed via a processive mechanism in which stereochemistry and oxidation state are adjusted following each consecutive condensation in the chain elongation process.; In an effort to isolate 10-deoxymethynolide (10-dMET), the parent aglycone of methymycin and neomethymycin, we screened several P-450 monooxygenase inhibitors for the ability to induce accumulation of 10-dMET without compromising the viability of the producing organism. We report the successful isolation and characterization of 10-dMET produced by fermenting cultures of Streptomyces venezuelae ATCC 15439 grown in the presence of xanthotoxin.; A point of conjecture in the biosynthesis of erythromycin A (ErA) has been whether the C-12 hydroxylase would operate on both erythromycin B (ErB) and erythromycin D (ErD) at the pathway branch introduced by the SAM dependent methylase EryG. Stassi et al. (Stassi, D., Donadio, S., Staver, M. J., & Katz, L. (1993) J. Bacteriol., 175, 182-189) cloned the gene, designated eryK, which encodes the P-450 monooxygenase responsible for C-12 hydroxylation in the biosynthesis of ErA. We report the heterologous overexpression of EryK in Escherichia coli as insoluble inclusion bodies; the solubilization, refolding and reconstitution of active holo-EryK; and kinetic confirmation of EryK's 1200 to 2000-fold preference for ErD over the alternative C-12 hydroxylase substrate ErB. Our conclusions support the long held assumption that ErB is a shunt metabolite in the erythromycin biosynthetic pathway.