Shafiee A, Motamedi H, Chen T
Department of Microbial Therapeutics and Molecular Genetics, Merck Research Laboratories, Rahway, NJ 07065.
Eur J Biochem. 1994 Oct 15;225(2):755-64. doi: 10.1111/j.1432-1033.1994.00755.x.
FK-506 is a macrolide antibiotic with immunosuppressant activity. Structurally, this compound contains three methylated hydroxyl groups at C13, C15 and C31. Previous biosynthetic studies using stable isotope-feeding experiments have established methionine as the source of the methyl for these methylated hydroxyl groups. Based on this information and also the availability of the 31-O-desmethylFK-506, a metabolic precursor for the biosynthesis of FK-506, a S-adenosyl-L-methionine-dependent enzyme assay was developed and the enzyme 31-O-desmethylFK-506 O:methyl-transferase was isolated from an extract of Streptomyces sp. MA 6858 and purified to near homogeneity. 31-O-DesmethylFK-506 O:methyltransferase is a monomeric protein with an apparent molecular mass of 30,000 Da and a pI of 4.4. The first 38 N-terminal amino acids have been sequenced and are H2N-SDVVETLRLPNGATVAHVNAGEAQFLYREIFTDRXYLRH. Functionally, This enzyme has a requirement for Mg2+ with an optimum temperature of 34 degrees C and a pH of 7.4 for full activity. Moreover, it catalyses the methylation of 31-O-desmethylimmunomycin as efficiently as its own natural substrate, 31-O-desmethylFK-506. Additionally, FKMT catalyzes the C31 transmethylation reaction of 13,31-O-bis-desmethyl-, 15,31-O-bisdesmethyl-, 13,15,31-O-trisdesmethyl- and 31-O-19,22-cyclic-hemiketalimmunomycins, which are all structural analogues of FK-506. The reaction is, however, completely blocked if the vicinal hydroxyl which is present at the C-32 position of the 31-O-desmethylFK-506 structure is replaced with azide, phosphate or other substituents. Finally, evidence is presented indicating the close similarity of FKMT and DIMT, a 31-O-desmethyl-immunomycin: O methyltransferase, previously isolated from a cell-free extract of Streptomyces hygroscopicus var ascomyceticus, an immunomycin (ascomycin/FK-520) producer.
FK-506是一种具有免疫抑制活性的大环内酯类抗生素。从结构上看，该化合物在C13、C15和C31处含有三个甲基化羟基。以往利用稳定同位素示踪实验进行的生物合成研究已确定甲硫氨酸是这些甲基化羟基中甲基的来源。基于这一信息以及FK-506生物合成的代谢前体31-O-去甲基FK-506的可得性，开发了一种依赖S-腺苷-L-甲硫氨酸的酶分析方法，并从链霉菌属MA 6858的提取物中分离出31-O-去甲基FK-506 O:甲基转移酶并纯化至接近均一。31-O-去甲基FK-506 O:甲基转移酶是一种单体蛋白，表观分子量为30,000 Da，pI为4.4。已对其N端前38个氨基酸进行了测序，序列为H2N-SDVVETLRLPNGATVAHVNAGEAQFLYREIFTDRXYLRH。在功能上，该酶需要Mg2+，最适温度为34℃，最适pH为7.4以实现完全活性。此外，它催化31-O-去甲基免疫霉素的甲基化反应，效率与它自身的天然底物31-O-去甲基FK-506相同。此外，FKMT催化13,31-O-双去甲基-、15,31-O-双去甲基-、13,15,31-O-三去甲基-和31-O-19,22-环半缩酮免疫霉素的C31甲基转移反应，这些都是FK-506的结构类似物。然而，如果31-O-去甲基FK-506结构中C-32位的邻位羟基被叠氮化物、磷酸盐或其他取代基取代，反应则会完全受阻。最后，有证据表明FKMT与DIMT密切相似，DIMT是一种先前从吸水链霉菌变株子囊菌（免疫霉素（子囊霉素/FK-520）的生产者）的无细胞提取物中分离出的31-O-去甲基免疫霉素：O甲基转移酶。
