Purification, characterization, and kinetic mechanism of S-adenosyl-L-methionine:macrocin O-methyltransferase from Streptomyces fradiae.
S-Adenosyl-L-methionine:macrocin O-methyltransferase catalyzes conversion of macrocin to tylosin, the terminal and main rate-limiting step of tylosin biosynthesis in Streptomyces fradiae. The O-methyltransferase was stabilized in vitro and purified to electrophoretic homogeneity. The purified enzyme had a molecular weight of 65,000 and consisted of two identical subunits of 32,000 with an isoelectric point of 4.5. The enzyme required Mg2+, Mn2+, or Co2+ for maximal activity and was catalytically optimal at pH 7.5-8.0 and 31 degrees C. The O-methyltransferase catalyzed the conversion of macrocin to tylosin at a stoichiometric ratio of 1:1. The enzyme also mediated conversion of lactenocin----desmycosin. The corresponding Vmax/Km ratios for the two analogous conversions were similar, and both enzymic conversions were susceptible to extensive competitive and noncompetitive inhibitions by macrolide metabolites. Steady-state kinetic studies for initial velocity, substrate analogue, and product inhibitions have allowed formulation of Ordered Bi Bi as the reaction mechanism for macrocin O-methyltransferase.
Bauer
N J
NJ
Biochemical Development Division, Eli Lilly and Company, Indianapolis, Indiana 46285.
Kreuzman
A J
AJ
Dotzlaf
J E
JE
Yeh
W K
WK
D000596:Amino Acids / Q000032:analysis; D055598:Chemical Phenomena; D002621:Chemistry; D003035:Cobalt / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007933:Leucomycins / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008780:Methyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D013302:Streptomyces / Q000201:enzymology*; D015645:Tylosin
D000596:Amino Acids; D007933:Leucomycins; D003035:Cobalt; D008345:Manganese; D008780:Methyltransferases; C032326:S-adenosyl-L-methionine-macrocin O-methyltransferase; D008274:Magnesium; D015645:Tylosin