Published on Web 07/28/2007
Kinetic Analysis of Teicoplanin Glycosyltransferases and Acyltransferase
Reveal Ordered Tailoring of Aglycone Scaffold to Reconstitute Mature
Teicoplanin
Annaleise R. Howard-Jones,§ Ryan G. Kruger,§,| Wei Lu,§,† Junhua Tao,‡ Catherine Leimkuhler,#,
Daniel Kahne,# and Christopher T. Walsh*,§
Department of Biological Chemistry & Molecular Pharmacology, HarVard Medical School, 240 Longwood AVe,
Boston, Massachusetts 02115, BioVerdant, Inc., 7330 Carroll Road, San Diego, California 92121, and
Department of Chemistry & Chemical Biology, HarVard UniVersity, Cambridge, Massachusetts 02138
Received May 18, 2007; E-mail: christopher_walsh@hms.harvard.edu
The glycopeptide antibiotics, teicoplanin, vancomycin, and
chloroeremomycin, are clinically important compounds used as a
treatment of last resort for methicillin-resistant Staphylococcus
aureus and other Gram-positive pathogens.1-3 Clinically adminis-
tered teicoplanin comprises a naturally occurring mixture of five
differentially acylated scaffolds, including teicoplanin A2-3 1
(Figure 1).9 The heptapeptide scaffolds of these molecules are
assembled by nonribosomal peptide synthetases,4 with extensive
oxidative crosslinking giving rigidity to the antibiotic cores.5,6
Figure 1. Chemical structure of glycopeptide, teicoplanin A2-3 1. Positions
4 and 6 are indicated in red.
Importantly, after release from the enzymatic assembly lines, all
of the family members are modified through glycosylation, and
aminoglucose)-AGT 3, and 35-fold lower kcat/Km relative to the
parameters for 4-GlcNAc-AGT 4 (Table 1). Hence, tGtfA prefers
a scaffold monoglycosylated at position 4, but is fairly indiscrimi-
some through acylation,7 modifications that are important in
conferring biological activity.8 We have been interested in char-
acterizing the postassembly line-tailoring processes of glycopeptides
nate regarding the N-acylation state of the sugar moiety at this
to understand the enzymatic properties of the respective catalysts
position (Scheme 1).
and to construct frameworks for the rational design of new
The AGT scaffold preglycosylated at position 6 (6-GlcNAc-
analogues.
AGT) is not accepted by tGtfB at all, regardless of whether UDP-
GlcNAc, UDP-2-aminoglucose, or UDP-(N-decanoyl)-2-aminoglu-
cose is used as the glycosyl donor (data not shown). However, AGT
2 is readily accepted by this enzyme (Table 1). Overall, these data
suggest that tGtfB acts first on the naked aglycone 2 to glycosylate
at position 4. tGtfA acts subsequently to condense a second GlcNAc
moiety onto either the 4-GlcNAc-AGT 4 or 4-(2′-aminoglucose)-
AGT 3 scaffolds at position 6, giving bisglycosylated heptapeptides
5 and 6 (Scheme 1).
It has been shown that the vancomycin and chloroeremomycin
glycosyltransferases, GtfB, GtfC, GtfD, and GtfE and the acyl-
transferase, aAtf, will accept both vancomycin and teicoplanin
The glycosyltransferases, tGtfA and tGtfB, are able to act
separately on the teicoplanin aglycone 2 and also in tandem to
produce the bisglycosylated heptapeptide 6.6 The acyltransferase,
tAtf, is known to mediate condensation of an acyl CoA with the
teicoplanin scaffold glycosylated at position 46,10 and also with the
UDP sugar, UDP-2-aminoglucose.6 For transfer to the glycosylated
aglycone 3, a range of acyl chains could successfully be transferred,
with decanoyl CoA giving the highest activity;10 decanoyl CoA
has hence been used as the preferred acyl donor throughout this
work, thus constituting the pathway to teicoplanin A2-3 1. In the
absence of any kinetic data, however, it was impossible to postulate
which would be the preferred acyl and glycosyl acceptors for these
scaffolds.13,14,10 In light of this promiscuity, particularly in the related
enzymes in vivo. Building on our prior work to establish the kinetic
Gtfs, it is interesting that neither tGtfA nor tGtfB can catalyze
order of action of the glycosyltransferases of the vancomycin and
glycosylation of the vancomycin aglycone core (data not shown).
chloroeremomycin families,11,12 we report here the first detailed
Both teicoplanin Gtfs are highly selective for their cross-linked
kinetic characterization of the teicoplanin glycosyltransferases
peptide scaffold, implying that their active sites are capable of
(tGtfA and tGtfB) and acyltransferase (tAtf).
sensing chemical groups distal to the reactive functionalities as well
Initially, we sought to determine the relative order of addition
as the overall conformation of the glycosyl acceptor.
of the glycosyl moieties at positions 6 and 4 of the aglycone, by
tGtfB is relatively promiscuous in terms of the sugar donor that
the action of tGtfA and tGtfB, respectively. Thorough kinetic
it will utilize: UDP-GlcNAc, UDP-2-aminoglucose, and UDP-(N-
decanoyl)-2-aminoglucose are all accepted with similar catalytic
analysis revealed that, although tGtfA is able to catalyze addition
of UDP-GlcNAc to the aglycone 2, it does so with 56-fold lower
efficiencies, effecting transfer onto the AGT aglycone 2 (Table 1).
catalytic efficiency relative to addition of the same sugar to 4-(2′-
The concentration of UDP-GlcNAc is likely much higher in the
cell relative to the other glycosyl donors, so the probable glyco-
§ Harvard Medical School.
sylation pathway in vivo involves tGtfB-mediated glycosylation
with UDP-GlcNAc. The teicoplanin biosynthetic cluster contains
a dedicated deacylase,15 which has been shown to carry out
deacetylation of 4-GlcNAc-AGT 4 to afford 4-(2′-aminoglucose)-
AGT 3.15 Deacylated scaffold 3 or 5 would be required to enable
the action of acyltransferase tAtf (Scheme 1).
| Current address: GlaxoSmithKline Pharmaceuticals UP1345, 1250 S. Colle-
geville Road, Collegeville, PA 19426.
† Current address: Merck Research Laboratories-Boston, BMB 10-120, 33
Avenue Louis Pasteur, Boston, MA 02115.
‡ BioVerdant Inc.
# Harvard University.
Current address: Department of Molecular and Cellular Biology, Harvard
University, 7 Divinity Ave, Bauer 308, Cambridge, MA 02138.
9
10082
J. AM. CHEM. SOC. 2007, 129, 10082-10083
10.1021/ja0735857 CCC: $37.00 © 2007 American Chemical Society
Howard-Jones, Annaleise R.
