THE BIOSYNTHETIC GENE-CLUSTER FOR THE MACROLACTONE RING OF THE IMMUNOSUPPRESSANT FK506

Biosynthesis of the macrolactone ring of FK506 involves 10 elongation cycles that mechanistically resemble the steps in fatty acid synthesis . Sequencing of a 40-kb DNA segment of the FK506 gene cluster from Str eptomyces sp. MA6548 has revealed two additional polyketide synthases (PKS) genes fkbB and fkbC which lie upstream of fkbA, a PKS gene recen tly shown to be responsible for the last four condensation steps of th e FK506 biosynthesis [Motamedi, H., Cai, S. J., Shafiee, A. & Elliston , K. O. (1997) fur: J. Biochem. 244, 74-80]. fkbB and fkbC are contigu ous and encode respectively, the first (790129 Dal and the second (374 438 Dal components of the FK506 polyketide synthase, a complex of thre e multidomain polypeptides. The predicted domain structures of FkbB an d FkbC are analogous to that of FkbA and comprise 30 fatty-acid-syntha se(FAS)-like domains arranged in 6 modules. Each module performs a spe cific extension cycle in the assembly of the carbon skeleton of the FK 506 macrolactone ring. The component activities for the initiation of the polyketide chain consisting of a dihydrocyclohexenylcarbonyl coenz yme A (CoA) synthetase and a dihydrocyclohexenylcarbonyl CoA reductase required for the formation of the dihydrocyclohexylcarbonyl CoA start er unit and an acyl-carrier-protein to which the starter unit is ancho red and translocated to the appropriate site on the PKS multienzyme ar e located at the N-terminal region of the FkbB polypeptide. A third ge ne, fkbL, lies at one end of the cluster and encodes lysine cyclodeami nase which catalyzes a-deamination and cyclization of the lysine into pipecolate. A fourth gene fkbP located at the other end of the sequenc e reported here encodes a peptide synthetase required for the activati on and incorporation of the pipecolate moiety into the completed acyl chain. Finally the cluster carries a gene, fkbO, whose product is pres umed to carry out a post-polyketide oxidation step of the FK506 marocy cle.