Characterization of rhodosaminyl transfer by the AknS/AknT glycosylation complex and its use in reconstituting the biosynthetic pathway of aclacinomycin A.
The tetracyclic core of anthracycline natural products with antitumor activity such as aclacinomycin A are tailored during biosynthesis by regioselective glycosylation. We report the first synthesis of TDP-L-rhodosamine and demonstrate that the glycosyltransferase AknS transfers L-rhodosamine to the aglycone to initiate construction of the side-chain trisaccharide. The partner protein AknT accelerates AknS turnover rate for L-rhodosamine transfer by 200-fold. AknT does not affect the Km but rather affects the kcat. Using these data, we propose that AknT causes a conformational change in AknS that stabilizes the transition state and ultimately enhances transfer. When the subsequent glycosyltransferase AknK and its substrate TDP-L-fucose are also added to the aglycone, the disaccharide and low levels of a fully reconstituted trisaccharide form of aclacinomycin are observed.
Leimkuhler
Catherine
C
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
Fridman
Micha
M
Lupoli
Tania
T
Walker
Suzanne
S
Walsh
Christopher T
CT
Kahne
Daniel
D
D015250:Aclarubicin / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D000970:Antineoplastic Agents / Q000737:chemistry; D006031:Glycosylation; D016695:Glycosyltransferases / Q000737:chemistry* / Q000378:metabolism*; D006595:Hexosamines / Q000378:metabolism*; D007700:Kinetics; D018942:Macrolides / Q000737:chemistry*; D015394:Molecular Structure; D013379:Substrate Specificity
D000970:Antineoplastic Agents; D006595:Hexosamines; D018942:Macrolides; D015250:Aclarubicin; D016695:Glycosyltransferases