Motamedi H, Shafiee A, Cai S J, Streicher S L, Arison B H, Miller R R
Department of Natural Products Drug Discovery, Merck Research Laboratories, Rahway, New Jersey 07065, USA.
J Bacteriol. 1996 Sep;178(17):5243-8. doi: 10.1128/jb.178.17.5243-5248.1996.
FK506 and FK520 are 23-membered macrocyclic polyketides with potent immunosuppressive and antifungal activities. The gene encoding 31-O-demethyl-FK506 methyltransferase, fkbM, was isolated from Streptomyces sp. strains MA6858 and MA6548, two FK506 producers, and Streptomyces hygroscopicus subsp. ascomyceticus, an FK520 producer. The nucleotide sequence of the fkbM gene revealed an open reading frame encoding a polypeptide of 260 amino acids. Disruption of fkbM in Streptomyces sp. strain MA6548 yielded a mutant that produced 31-O-demethyl-FK506, confirming the involvement of the isolated genes in the biosynthesis of FK506 and FK520. Heterologous expression of fkbM in Streptomyces lividans established that fkbM encodes an O-methyltransferase catalyzing the methylation of the C-31 hydroxyl group of 31-O-demethyl-FK506 and FK520. A second open reading frame, fkbD, was found upstream of fkbM in all three aforementioned species and was predicted to encode a protein of 388 residues that showed a strong resemblance to cytochrome P-450 hydroxylases. Disruption of fkbD had a polar effect on the synthesis of the downstream fkbM gene product and resulted in the formation of 9-deoxo-31-O-demethyl-FK506. This established the product of fkbD as the cytochrome P-450 9-deoxo-FK506 hydroxylase, which is responsible for hydroxylation at position C-9 of the FK506 and FK520 macrolactone ring.
FK506和FK520是具有强大免疫抑制和抗真菌活性的23元大环聚酮化合物。编码31 - O - 去甲基 - FK506甲基转移酶的基因fkbM，是从两种FK506产生菌链霉菌属菌株MA6858和MA6548，以及一种FK520产生菌吸水链霉菌子囊菌亚种中分离得到的。fkbM基因的核苷酸序列显示出一个编码260个氨基酸多肽的开放阅读框。链霉菌属菌株MA6548中fkbM的破坏产生了一个产生31 - O - 去甲基 - FK506的突变体，证实了分离出的基因参与FK506和FK520的生物合成。fkbM在变铅青链霉菌中的异源表达表明，fkbM编码一种O - 甲基转移酶，催化31 - O - 去甲基 - FK506和FK520的C - 31羟基的甲基化。在上述所有三个物种中，在fkbM的上游发现了第二个开放阅读框fkbD，预计它编码一个388个残基的蛋白质，该蛋白质与细胞色素P - 450羟化酶有很强的相似性。fkbD的破坏对下游fkbM基因产物的合成有极性影响，并导致9 - 脱氧 - 31 - O - 去甲基 - FK506的形成。这确定了fkbD的产物为细胞色素P - 450 9 - 脱氧 - FK506羟化酶，它负责FK506和FK520大环内酯环C - 9位的羟基化。
