ENZYMOLOGY OF FK-506 BIOSYNTHESIS - PURIFICATION AND CHARACTERIZATIONOF 31-O-DESMETHYLFK-506 O-METHYLTRANSFERASE FROM STREPTOMYCES SP MA6858

FK-506 is a macrolide antibiotic with immunosuppressant activity. Stru cturally, this compound contains three methylated hydroxyl groups at C 13, C15 and C31. Previous biosynthetic studies using stable isotope-fe eding experiments have established methionine as the source of the met hyl for these methylated hydroxyl groups. Based on this information an d also the availability of the 31-O-desmethylFK-506, a metabolic precu rsor for the biosynthesis of FK-506, a S-adenosyl-L-methionine-depende nt enzyme assay was developed and the enzyme 31-O-desmethylFK-506 O:me thyltransferase was isolated from an extract of Streptomyces sp. MA 68 58 and purified to near homogeneity. 31-O-DesmethylFK-506 O:methyltran sferase is a monomeric protein with an apparent molecular mass of 3000 0 Da and a pI of 4.4. The first 38 N-terminal amino acids have been se quenced and are H2N-SDVVETLRLPNGATVAHVNAGEAQFLYREIFTDRXYLRH. Functiona lly, this enzyme has a requirement for Mg2+ with an optimum temperatur e of 34 degrees C and a pH of 7.4 for full activity. Moreover, it cata lyses the methylation of 31-O-desmethylimmunomycin as efficiently as i ts own natural substrate, 31-O-desmethylFK-506. Additionally, FKMT cat alyzes the C31 transmeth ylation reaction of 13,31-O-bis-desmethyl-, 1 5,31-O-bisdesmethyl-, 13,15,31-O-trisdesmethyl- and 31-O-19,22-cyclic- hemiketalimmunomycins, which are all structural analogues of FK-506. T he reaction is, however, completely blocked if the vicinal hydroxyl wh ich is present at the C-32 position of the 31-O-desmethylFK-506 struct ure is replaced with azide, phosphate or other substituents. Finally, evidence is presented indicating the close similarity of FKMT and DIMT , a 31-O-desmethylimmunomycin:O methyltransferase, previously isolated from a cell-free extract of Streptomyces hygroscopicus var ascomyceti cus, an immunomycin (ascomycin/FK-520) producer.