AknK is an L-2-deoxyfucosyltransferase in the biosynthesis of the anthracycline aclacinomycin A.
The antitumor drug aclacinomycin A is a representative member of the anthracycline subgroup that contains a C(7)-O-trisaccharide chain composed of L-2-deoxysugars. The sugar portion of the molecule, which greatly affects its biological activity, is assembled by dedicated glycosyltransferases; however, these enzymes have not been well-studied. Here we report the heterologous expression and purification of one of these enzymes, AknK, as well as the preparation of dTDP-L-2-deoxysugar donors, dTDP-L-2-deoxyfucose and dTDP-L-daunosamine, and the monoglycosyl aglycone, rhodosaminyl aklavinone. Our experiments show that AknK catalyzes the addition of the second sugar to the chain, using dTDP-L-2-deoxyfucose and rhodosaminyl aklavinone, to create the L-2-deoxyfucosyl-L-rhodosaminyl aklavinone. AknK also accepts an alternate dTDP-L-sugar, dTDP-L-daunosamine, and other monoglycosylated anthracyclines, including daunomycin, adriamycin, and idarubicin, to build alternate disaccharides on variant anthracycline backbones. Remarkably, AknK also catalyzes a tandem addition of a second L-2-deoxyfucosyl moiety, albeit with reduced activity, to the natural disaccharide chain to produce L-deoxyfucosyl-L-deoxyfucosyl-L-rhodosaminyl aklavinone, a variant of the natural aclacinomycin A. These results demonstrate that AknK may be a useful enzyme for the chemoenzymatic synthesis of anthracycline variants.
Lu
Wei
W
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Leimkuhler
Catherine
C
Oberthür
Markus
M
Kahne
Daniel
D
Walsh
Christopher T
CT
D015250:Aclarubicin / Q000031:analogs & derivatives / Q000096:biosynthesis*; D002384:Catalysis; D003001:Cloning, Molecular; D005643:Fucose / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000737:chemistry; D005647:Fucosyltransferases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D006031:Glycosylation; D006595:Hexosamines / Q000096:biosynthesis / Q000737:chemistry; D009279:Naphthacenes / Q000138:chemical synthesis; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D013942:Thymine Nucleotides / Q000138:chemical synthesis; D014312:Trisaccharides / Q000737:chemistry
D006595:Hexosamines; D009279:Naphthacenes; D013942:Thymine Nucleotides; D014312:Trisaccharides; C027832:aklavinone; C007217:daunosamine; D005643:Fucose; D015250:Aclarubicin; D005647:Fucosyltransferases