Characterization of glycosyltransferase DesVII and its auxiliary partner protein DesVIII in the methymycin/picromycin biosynthetic pathway.
The in vitro characterization of the catalytic activity of DesVII, the glycosyltransferase involved in the biosynthesis of the macrolide antibiotics methymycin, neomethymycin, narbomycin, and pikromycin in Streptomyces venezuelae, is described. DesVII is unique among glycosyltransferases in that it requires an additional protein component, DesVIII, for activity. Characterization of the metabolites produced by a S. venezuelae mutant lacking the desVIII gene confirmed that desVIII is important for the biosynthesis of glycosylated macrolides but can be replaced by at least one of the homologous genes from other pathways. The addition of recombinant DesVIII protein significantly improves the glycosylation efficiency of DesVII in the in vitro assay. When affinity-tagged DesVII and DesVIII proteins were coproduced in Escherichia coli, they formed a tight (αβ)(3) complex that is at least 10(3)-fold more active than DesVII alone. The formation of the DesVII/DesVIII complex requires coexpression of both genes in vivo and cannot be fully achieved by mixing the individual protein components in vitro. The ability of the DesVII/DesVIII system to catalyze the reverse reaction with the formation of TDP-desosamine was also demonstrated in a transglycosylation experiment. Taken together, our data suggest that DesVIII assists the folding of DesVII during protein production and remains tightly bound during catalysis. This requirement must be taken into consideration in the design of combinatorial biosynthetic experiments with new glycosylated macrolides.
Borisova
Svetlana A
SA
Division of Medicinal Chemistry, College of Pharmacy, and Department of Chemistry and Biochemistry, University of Texas, Austin, Texas 78712, USA.
Liu
Hung-Wen
HW
D000606:Amino Sugars; D000900:Anti-Bacterial Agents / Q000096:biosynthesis* / Q000378:metabolism; D053898:Biosynthetic Pathways; D004926:Escherichia coli / Q000378:metabolism; D006031:Glycosylation; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism*; D018942:Macrolides / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013302:Streptomyces / Q000235:genetics / Q000378:metabolism
D000606:Amino Sugars; D000900:Anti-Bacterial Agents; D018942:Macrolides; D011506:Proteins; D011994:Recombinant Proteins; C010488:narbomycin; C008517:methymycin; C008594:neomethymycin; C110125:desosamine; D016695:Glycosyltransferases; C003145:picromycin