Studies Of Pyran Rings Biosynthesis Of FR901464

FR901464 represents potent anticancer activity by inhibiting the spliceosorae. It is biosynthesized by AT-less PKSs hybridized with a nonribosomal peptide synthetase (NRPS). It contains two highly fimctionalized tetrahydropyran rings, A-ring and B-ring. Based on the bioinfonnatics analysis, we supposed that the formation of the A-ring is catalyzed by a pyran synthase domain (named Fr9F-PSA) or an independent domain (FR9Q) through Michael addition reaction, and the formation of the B-ring is is catalyzed by FR9I-PSB.In order to study the biosynthesis mechanism of A-ring, we first synthesized a simple analog substrate. But no reaction was detected when we incubated it with FR9F-PSA or FR9Q. A more alike substrate, which consists of four diastereoisomers, was synthesized and tested by HPLC and LC-MS. Only Fr9F-PSA converted one of the isomers to the product. The structure and configuration of the cyclized product was resolved by NOSEY, and is consistent with the A-ring of FR901464 and the structure of the the transformed substrate was deduced by the product. So we concluded that the formation of A-ring was catalyzed by FR9F-PSA. The activity abolished by H34A mutant suggests that His34 is essential for FR9F-PSA activity and may serve as general base to abstract proton from substrate.FR9I-PSA cannot catalyze the transformation of all the substrates used in this article into the corresponding cyclized products.These results showed that Fr9F-PSA is a stringent substrate-and stereo-selective pyran synthase domain which responsible for the formation of A-ring of FR901464. And the FR9I-PSA related to the formation of B-ring may also be an enzyme with high substrate selectivity.