Direct evidence that the rifamycin polyketide synthase assembles polyketide chains processively

The assembly of the polyketide backbone of rifamycin B on the type I rifamy cin polyketide synthase (PKS), encoded by the rifA-rifE genes, is terminate d by the product of the rifF gene, an amide synthase that releases the comp leted undecaketide as its macrocyclic lactam. Inactivation of rifE gives a rifamycin B nonproducing mutant that still accumulates a series of linear p olyketides ranging from the tetra- to a decaketide, also detected in the wi ld type, demonstrating that the PKS operates in a processive manner. Disrup tions of the rifD module 8 and rifE module 9 and module 10 genes also resul t in accumulation of such linear polyketides as a consequence. of premature termination of polyketide assembly. Whereas the tetraketide carries an unm odified aromatic chromophore, the penta- through decaketides have undergone oxidative cyclization to the naphthoquinone, suggesting that this modifica tion occurs during, not after, PKS assembly. The structure of one of the ac cumulated compounds together with O-18 experiments suggests that this oxida tive cyclization produces an 8-hydroxy-7,8-dihydronaphthoquinone structure that, after the stage of proansamycin X, is dehydrogenated to an 8-hydroxyn aphthoquinone.