Published on Web 08/08/2007
Characterization of Rhodosaminyl Transfer by the AknS/AknT
Glycosylation Complex and Its Use in Reconstituting the
Biosynthetic Pathway of Aclacinomycin A
Catherine Leimkuhler,# Micha Fridman, Tania Lupoli, Suzanne Walker,
Christopher T. Walsh, and Daniel Kahne*
Contribution from the Department of Chemistry and Chemical Biology, HarVard UniVersity,
Cambridge, Massachusetts 02138, Department of Biological Chemistry and Molecular
Pharmacology, and Department of Microbiology and Molecular Genetics, HarVard Medical
School, 200 Longwood AVenue, Boston, Massachusetts 02115
Received April 25, 2007; E-mail: kahne@chemistry.harvard.edu
Abstract: 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.
Introduction
sequentially attaches TDP-2-deoxy-L-fucose (7) and TDP-L-
rhodinose (8), producing first 2-deoxy-fucosyl-rhodosaminyl-
AknS is a glycosyltransferase (Gtf) that is involved in the
biosynthesis of aclacinomycin A (1, Figure 1). Aclacinomycin
A is a member of the anthracyclines, a class of microbial
secondary metabolites produced by Streptomyces galilaeus with
antitumor activity. Aclacinomycin A, along with other members
of its class, including daunomycin (2) and adriamycin (3), is
used in the clinic to treat various cancers.1 The mechanism of
action of these chemotherapeutics is reported to be induction
of apoptosis following binding to double-stranded DNA frag-
ments.2 All anthracyclines contain a conserved tetracyclic
aglycone, 7,8,9,10-tetrahydro-5,12-napthacenequinone, with a
mono- to trisaccharide moiety attached to the C7-OH.3 The
trisaccharide of aclacinomycin A, which consists of three
different deoxy sugars, L-rhodosoamine, 2-deoxy-L-fucose, and
L-cinerulose A, has been shown to play a key role in binding to
the minor groove of target DNA sequences.4-6 The trisaccharide
is assembled by two Gtfs, AknS, which attaches the first
carbohydrate moiety, TDP-L-rhodosamine (5, Figure 2), to the
(4, Figure 2) to yield rhodosaminyl-aklavinone (6). AknK then
aklavinone (9) and then the trisaccharide, aclacinomycin A (3).7
The turnover of AknS is substantially increased by the
addition of an accessory protein, AknT,8 which is encoded by
a gene (aknT) found directly upstream of the aknS gene.9 A
few other Gtfs involved in the biosynthesis of aminosugar-
containing macrolides have also been found to require accessory
proteins for good activity: DesVII, which is involved in the
biosynthesis of narbomycin and EryCIII, which is involved in
the biosynthesis of erythromycin.10,11 How these accessory
proteins accelerate glycosyltransfer is not known. The AknS/
AknT system is ideal for addressing this issue because the
aglycon substrate is chromogenic, which facilitates kinetic
analysis of the glycosylation reaction. Here we report that AknT
accelerates glycosyltransfer by increasing kcat by 2 orders of
magnitude; it has no effect on substrate binding.8 We propose
that AknT facilitates a conformational change in AknS that
stabilizes the transition state.
Experimental Section
Materials. AknS, AknT, and AknK were overexpressed and purified
as previously described.7,8 TDP-daunosamine and TDP-2-deoxy-L-
# Current address: Department of Molecular and Cellular Biology,
Harvard University, Bauer 307, 7 Divinity Avenue, Cambridge, MA 02138.
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Rich, A.; Wang, A. Biochemistry 1990, 29, 2538-2549.
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S. J. Am. Chem. Soc. 2005, 127, 14128-14129.
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J. AM. CHEM. SOC. 2007, 129, 10546-10550
10.1021/ja072909o CCC: $37.00 © 2007 American Chemical Society