Two distinctive O-methyltransferases catalyzing penultimate and terminal reactions of macrolide antibiotic (tylosin) biosynthesis. Substrate specificity, enzyme inhibition, and kinetic mechanism.
S-Adenosyl-L-methionine:demethylmacrocin O-methyltransferase catalyzes the conversion of demethylmacrocin to macrocin as the penultimate step of tylosin biosynthesis in Streptomyces fradiae. The O-methyltransferase was purified to electrophoretic homogeneity by a conventional chromatographic procedure. The purified enzyme appears to be trimeric with a molecular weight of 122,000-126,000 and a subunit size of 42,000. Its isoelectric point was 6.0. The enzyme required Mg2+ for maximal activity and was catalytically optimal at pH 7.8-8.5 and 42 degrees C. The O-methyltransferase catalyzed conversion of demethylmacrocin to macrocin at a stoichiometric ratio of 1:1. The O-methyltransferase also mediated conversion of demethyllactenocin----lactenocin. The corresponding Vmax/Km ratios for the two analogous conversions varied only slightly. Both enzymic conversions were susceptible to an extensive and identical range of metabolic inhibitions. Steady-state kinetic studies for initial velocity, substrate analogue, and product inhibitions are consistent with Ordered Bi Bi as the reaction mechanism of demethylmacrocin O-methyltransferase. Except for an identical kinetic mechanism, demethylmacrocin O-methyltransferase can be readily differentiated from macrocin O-methyltransferase by its physical and catalytic properties as well as metabolic inhibitions.
Kreuzman
A J
AJ
Biochemical Development Division, Eli Lilly and Company, Indianapolis, Indiana 46285.
Turner
J R
JR
Yeh
W K
WK
D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D007933:Leucomycins / Q000096:biosynthesis*; D008274:Magnesium / Q000378:metabolism; D008780:Methyltransferases / Q000378:metabolism*; D008970:Molecular Weight; D013302:Streptomyces / Q000201:enzymology; D013379:Substrate Specificity; D015645:Tylosin
D007933:Leucomycins; D008780:Methyltransferases; C032326:S-adenosyl-L-methionine-macrocin O-methyltransferase; C057398:demethylmacrocin O-methyltransferase; D008274:Magnesium; D015645:Tylosin