The biosynthetic gene cluster for the macrolactone ring of the immunosuppressant FK506

Abstract

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 Streptomyces sp. MA6548 has revealed two additional polyketide synthases (PKS) genes fkbB and fkbC which lie upstream of fkbA, a PKS gene recently shown to be responsible for the last four condensation steps of the FK506 biosynthesis [Motamedi, H., Cai, S. J., Shafiee, A. & Elliston, K. O. (1997) Eur. J. Biochem. 244, 74Ϫ80]. fkbB and fkbC are contiguous and encode respectively, the first (790 129 Da) and the second (374 438 Da) components of the FK506 polyketide synthase, a complex of three multidomain polypeptides. The predicted domain structures of FkbB and FkbC are analogous to that of FkbA and comprise 30 fatty-acid-synthase(FAS)-like domains arranged in 6 modules. Each module performs a specific extension cycle in the assembly of the carbon skeleton of the FK506 macrolactone ring. The component activities for the initiation of the polyketide chain consisting of a dihydrocyclohexenylcarbonyl coenzyme A (CoA) synthetase and a dihydrocyclohexenylcarbonyl CoA reductase required for the formation of the dihydrocyclohexylcarbonyl CoA starter unit and an acyl-carrier-protein to which the starter unit is anchored and translocated to the appropriate site on the PKS multienzyme are located at the N-terminal region of the FkbB polypeptide. A third gene, fkbL, lies at one end of the cluster and encodes lysine cyclodeaminase which catalyzes A-deamination and cyclization of the lysine into pipecolate. A fourth gene fkbP located at the other end of the sequence reported here encodes a peptide synthetase required for the activation and incorporation of the pipecolate moiety into the completed acyl chain. Finally the cluster carries a gene, fkbO, whose product is presumed to carry out a post-polyketide oxidation step of the FK506 marocycle.

Biosynthesis of the immunosuppressants FK506, FK520, and rapamycin involves a previously undescribed family of enzymes acting on chorismate

“…Initial comparison of the biosynthetic gene clusters for rapamycin (15)(16)(17), FK506 (26,27), and FK520 (28) did not reveal obvious candidate genes encoding the enzymes of such a pathway. However, detailed genetic analysis has since revealed an essential role for rapK in the production of DHCHC during rapamycin biosynthesis (29).…”

A multifunctional polyketide–peptide synthetase essential for albicidin biosynthesis in Xanthomonas albilineans The GenBank accession number for the sequence determined in this work is AF239749.

“…( Aparicio et al, 1996 ;Motamedi & Shafiee, 1998 ;Tang et al, 1998 ;Duitman et al, 1999). It may be possible to determine the substrate specificity of the AL domain by heterologous expression and purification of the module 1 protein containing AL plus ACP1 domains, and ATPpyrophosphate exchange assays of the purified fusion protein with potential substrates, as applied to NRPS internal A domains (Guenzi et al, 1998 ;Stachelhaus et al, 1998Stachelhaus et al, , 1999.…”

Elucidating the Substrate Specificity and Condensation Domain Activity of FkbP, the FK520 Pipecolate-Incorporating Enzyme

“…Ni-NTA chromatography resin was purchased from Qiagen. [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]Acetyl-CoA and Amplify were obtained from Amersham Biosciences. Sodium [ 32 P]pyrophosphate was purchased from Perkin-Elmer.…”