Properties of S-adenosyl-L-methionine:macrocin O-methyltransferase in extracts of Streptomyces fradiae strains which produce normal or elevated levels of tylosin and in mutants blocked in specific O-methylations

An efficient assay for S-adenosyl-L-methionine:macrocin O-methyltransferase, the enzyme which carries out the terminal step in tylosin biosynthesis, is described. Macrocin O-methyltransferase requires Mg2+ and S-adenosyl-L-methionine for activity, has a temperature optimum of about 31 degrees C, and has a pH optimum of 7.5 to 8.2. Macrocin O-methyltransferase specifically converts macrocin to tylosin by O-methylation of the 3" ' position of macrocin. In vitro methylation studies with extracts from a tylosin-producing Streptomyces fradiae strain and from mutant strains blocked in 2" '- or 3" '-O-methylations indicated that: (i) the 2" '- and 3" '-O-methylations occur after 6-deoxy-D-allose is attached to the macrolide ring; (ii) the 2" '- and 3" '-O-methylations are carried out by separate enzymes; and (iii) the 2" '-O-methylation precedes the 3" '-O-methylation. Macrocin O-methyltransferase was inhibited by high levels of its substrate, macrocin, by its product, tylosin, and by other tylosin analogs which contained mycinose or demethyl analogs of mycinose. Macrocin O-methyltransferase was produced early in the tylosin fermentation cycle by S. fradiae and preceded the onset of rapid tylosin biosynthesis by about 24 h. The enzyme specific activity reached maximum at about 72 h and then slowly declined. A mutant strain of S. fradiae selected for increased tylosin production synthesized macrocin O-methyltransferase more rapidly and accumulated a higher enzyme specific activity than a wild-type strain.