Direct evidence that the rifamycin polyketide synthase assembles polyketide chains processively

Abstract

The assembly of the polyketide backbone of rifamycin B on the type I rifamycin polyketide synthase (PKS), encoded by the rifA-rifE genes, is terminated by the product of the rifF gene, an amide synthase that releases the completed undecaketide as its macrocyclic lactam. Inactivation of rifF gives a rifamycin B nonproducing mutant that still accumulates a series of linear polyketides ranging from the tetra-to a decaketide, also detected in the wild type, demonstrating that the PKS operates in a processive manner. Disruptions of the rifD module 8 and rifE module 9 and module 10 genes also result in accumulation of such linear polyketides as a consequence of premature termination of polyketide assembly. Whereas the tetraketide carries an unmodified aromatic chromophore, the penta-through decaketides have undergone oxidative cyclization to the naphthoquinone, suggesting that this modification occurs during, not after, PKS assembly. The structure of one of the accumulated compounds together with 18 O experiments suggests that this oxidative cyclization produces an 8-hydroxy-7,8-dihydronaphthoquinone structure that, after the stage of proansamycin X, is dehydrogenated to an 8-hydroxynaphthoquinone.

Identification of tailoring genes involved in the modification of the polyketide backbone of rifamycin B by Amycolatopsis mediterranei S699

“…However, mutant strain MT0031ABH, in which a 21 kb fragment of the post-PKS modification genes has been deleted, did not accumulate proansamycin X, but instead the tetraketides SY4b and desacetyl-SY4b were produced. These compounds have previously been isolated from knock-out mutants of the amide synthase RifF, as a result of premature release of the polyketide chain intermediates from the enzyme, starting from the tetraketide through the undecaketide (Stratmann et al, 1999;Yu et al, 1999). Structure identification of these intermediates showed that from the pentaketide onwards they contain a naphthalene ring, suggesting that the formation of the naphthalene moiety of rifamycin B takes place during the chain extension of the polyketide, specifically between the tetraketide and the pentaketide stage.…”

“…Based on results of the present study and others (Stratmann et al, 1999;Yu et al, 1999), it was clear that the core naphthalene ring moiety of rifamycin is constructed during the polyketide chain extension process. However, the fact that the reaction may require a catalytic enzyme, whose gene is located in the post-PKS modification region and functions in trans with the PKS, is rather unusual.…”

“…Instead, the mutant strain produced the tetraketide products SY4b and desacetyl-SY4b (the cyclic forms of P8/1-OG, Fig. 4B), which have previously been shown to be premature release products when the amide synthase (RifF) is inactivated (Stratmann et al, 1999;Yu et al, 1999). Negative-ion ESI-MS tandem analysis of desacetyl-SY4b gave fragment ions at m/z 246, 228,190,172,153,136, which are consistent with the expected fragmentation patterns for desacetyl-SY4b (Fig.…”

“…The amide synthase (RifF) catalyses the release of this linear product from the PKS complex and its cyclization via intramolecular amide formation. Inactivation of RifF results in the premature release of a series of polyketides of different chain length from the enzyme complex (Stratmann et al, 1999;Yu et al, 1999), and reveals that the formation of the naphthalene ring of rifamycin occurs during the chain extension. However, further studies are required to determine the mechanism and genes responsible for the construction of the naphthalene core motif.…”

ArylamineN-Acetyltransferase Responsible for Acetylation of 2-Aminophenols inStreptomyces griseus

“…However, recombinant RifF lacked any measurable INH acetylation activity (13). RifF catalyzes the release of the completed polyketide from the rifamycin type I polyketide synthase by intramolecular amide formation, yielding proansamycin X (26). Intramolecular amide formation appears to proceed by a reaction mechanism similar to that of the N acetylation by NAT.…”