The Role of Reversible Glycosyltransferases through the Biosynthesis of Calicheamicin
Maria Segun
Senior Comprehensive paper
Catholic University of America
Spring 2007
Abstract:
The purpose of this study is to evaluate the reversibility of glycosyltransferases
and to characterize their role in the making of anticancer drug Calicheamicin.
Glycosyltransferases are enzymes that catalyze the transfer of a monosaccharide unit
from an activated sugar phosphate to an acceptor such as alcohol. Glycosyl transfer
usually results in a monosaccharide glycoside, an oligosaccharide, or a polysaccharide.
Glycosyltransferases are generally unidirectional catalysts that can catalyze the transfer
of a glycosyl moiety with either a retention or inversion configuration. It has been shown
recently that Glycosyltransferases used in the biosynthetic pathway of Calicheamicin
readily catalyzes reversible reactions that allow for: 1) The transfer of sugars from one
natural backbone to the next, 2) the exchange of native natural glycosides with exogenous
carbohydrates, and 3) the synthesis of exotic NDP-sugars from glycosylated products.
Calicheamicins are a family of enidiyne antibiotics isolated from Micromonospora
echinospora that are used to target cancer cells by causing apoptosis. The newly found
reversibility of Glycosyltransferases allows for a simpler way of creating Calicheamicins
thus enhancing its performance and also, allowing for production of better performing
Calicheamicin variants.
References:
1) Zhang, C., Griffith, B., Quiang, F., Albermann, C., Fu, X., Lee, I., Lingjun, L.,
Thorson, J. Science 2006 313, 1291-1294.
2) Lee, M. Chem. Society 1987 109, 3463-3466.
3) Borman, S. Chemical engineering new 2006 84, 13-22.
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INTRODUCTION
Glycosylation is the process of adding saccharides to proteins and lipids using
different and specific glycosyltransferases (GT). This is one of the most important
processes involved in the synthesis of membrane and secreted proteins.¹ Glycosylation is
an enzyme directed process of two types: N-linked glycosylation to the amide nitrogen of
asparagines side chains and O-linked glycosylation to the hydroxyl oxygen of serine and
thereonine side chains.¹ N-linked glycosylation occurs in the Endoplasmic Reticulum
(ER), can be continued in the Golgi Apparatus (GA) and the process is initiated first with
a lipid-linked oligosaccharide precursor being synthesized (Figure 1). 2
Figure 1. Synthesis of Lipid-linked Oligosaccharide Precursor.3
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The precursor oligosaccharide is linked by a pyrophosphoryl group to dolichol. It is a
long (75-95 carbon atoms), highly hydrophobic polyisoprenoid lipid (Figure 2):
Figure 2. Structure of the Dol-P lipid used in constructing the dolichol oligosaccharide
precursor in N-linked oligosaccharide biosynthesis. The number of isoprene repeats (n)
varies between 15 and 19.1; 3
Dolichol is localized on the rough ER and the biosynthesis starts at the cytosolic
face of the ER. The process begins with 2 GlcNAc and 5 mannose residues being added
one at time to dolichol phosphate. The dolichol pyrophosphoryl oligosaccharide is then
flipped to the luminal face. Mannose or glucose residues are transferred from a nucleotide
sugar to dolichol on the cytosolic face of the ER. Then it is flipped and transferred to the
growing oligosaccharide. The carrier is then flipped back again to the cytosolic face.
Once this is done, the oligosaccharide precursor is transferred from the dolichol to an Asn
residue on the nascent polypeptide. Because of this process, glycosylation is seen as
occurring cotranslationally. The Asn residue must be in a sequence N-X-S/T where X
cannot be Pro or Asp 2 .The process is catalyzed by oligosaccharide protein transferase
which is made up of three subunits: two of them are ribophorins - ER transmembrane
proteins and the third subunit has the catalytic activity. 2
O-glycosylation is a stepwise addition of sugar residues directly to a polypeptide
chain and this process occurs in the GA 2. The process begins with the transfer of
GalNAc residue from UDP-GalNAc to the hydroxyl group of Ser or Thr residue. It is
catalyzed by N-acetylgalactosaminyltransferase. The UDP-GalNAc is synthesized from
4
UDP-(Glc). The protein is then moved to the trans-Golgi vesicles where the carbohydrate
chain is elongated. The specific GT adds the Gal residue to the GalNAc. The last step in
the biosynthesis of typical O-glycans is the additions of two N-acetylneuramic acid (sialic
acid) residues in the trans-Golgi reticulum. Figure 3 illustrates the process of Oglycosylation.
Figure 3. The Biosynthesis of O-linked Oligosaccharides.3
GTs work with a cofactor of either Mn or Mg at a pH range of 5-7 with the
catalytic sites facing the lumen of the various compartments of the GA and the sugar
nucleotide donors being made in the cytosol. GTs generally work in a linear sequential,
specific manner such that the oligosaccharide product of one enzyme yields a product
that serves as a preferred acceptor substrate for the subsequent action of other GTs.4 The
end result is a linear and/or branched polymer composed of monosaccharides linked to
5
one another (Figure 3). The nucleotide sugars used by the GTs during glycosylation
include GlcNAc, CMP-Sialic acid, Gal, and GDP-mannose (Figure 4).
Figure 3. Schematic of glycosyltransferase catalysis 3
Figure 4. Structures of four sugar nucleotides used in the biosynthesis of
oligosaccharides found in glycoproteins. 3
6
Due to recent research, it has been discovered that GTs that are normally thought
of as being unidirectional are bi-directional reversible catalysts. The newly found
reversibility of GTs were evaluated through the synthesis of anticancer drug
Calicheamicin. 4
History and structure of Calicheamicin
Calicheamicin (Figure 5) is an anticancer drug derived from the organism
Micromonospora echinospora. Calicheamicin works by binding to DNA, cleaving it into
tiny pieces and killing the tumor cells.
Figure 5. The structure of the nonchromoprotein enediyne calicheamicin. The enediyne
families are typically distinguished by either the number of carbons in the enediyne ring
(9-membered versus 10-membered) or by their association, or lack thereof, with an
apoprotein (chromoprotein versus nonchromoprotein) 5
The molecular structure of Calicheamicin works in three parts: (a) the 10
membered enidyne ring system which upon activation generates radicals via a Bergman
cyclization, which is an organic rearrangement reaction that takes place when an enyne is
heated in the presence of a suitable hydrogen donor, (b) the oligosaccharide domain
which recognizes and binds to the minor grove of DNA, thereby serving as a delivery
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system; and (c) the trisulfide moiety which serves as the initiation point for the cascade of
reactions leading ultimately to the diradical formation and the site-specific oxidative
double-strand slicing of the targeted DNA (Figure 6) 5.
Figure 6. Mechanistic action of Calicheamicin. 5
Recent in vitro and in vivo studies support the idea of Calicheamicin as a DNA-damaging
agent and also suggest that Calicheamicin favors cleavage at certain chromosomal sites4.
The downside however to Calicheamicin is that it is not tumor specific however,
scientists have been able to fix this problem by conjugating Calicheamicin to tumorspecific monoclonal antibodies.
Recent studies have shown that GTs used in the synthesis of Calicheamicin
catalyzes bi-directional reversible reactions. Specifically two GTs were tested:
Calicheamicin Cal G1 and Calicheamicin Cal G4 and they catalyzed the following three
reactions: (i) the synthesis of exotic NDP-sugars from glycosylated natural products, (ii)
the exchange of native natural-product glycosides with exogenous carbohydrates supplied
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as NDP-sugars, and (iii) the transfer of a sugar from one natural product backbone to a
distinct natural-product scaffold. More than 70 differently glycosylated Calicheamicin
variants were produced, which testifies to the reversibility of GTs. 4
The synthesis of exotic NDP-sugars from glycosylated natural products
Testing the idea of exotic sugar synthesis began by amplification, and expression
of the CalG1 gene in Escherichia coil. The recombinant GalG1 that resulted from this
process was purified and incubated with aglycon 1 (Figure 7A) and surrogate substrate
thymidine diphosphate (TDP)-ß-L-rhamnose. This reaction resulted in the formation of a
new product characterized as 2a by liquid chromatography-mass spectrometry (LC-MS)
and the aglycon as 1 (Figure 7D). The formation of product 2a was a result of
incubating 50μM of aglycon 1, 300μM of (TDP)-ß-L-rhamnose, and CalG1. This assay
was compared to the control in panel ii which consisted of 50 μM aglycon 2 (Figure 7A),
100 TDP, and CalG1. The control did not yield a product at 14.5 min (2a) as did the
earlier assay which further illustrates the formation of a new product.
The same reaction was followed using 9 additional TDP sugar substrates that
were converted to their corresponding Calcheamicin glycosides, 2b-2j (Figure 7A) in
conversions of 27% to 62%. The products of the CalG1 catalyzed reactions were
analyzed using LC-MS/MS. Again the fragmentation displayed is a result of an
attachment of the sugar to the aromatic ring of the substrate Calcheamicin. In support of
this result, a Calicheamicin library by CalG1-catalyzed sugar exchange (Figure 8) was
created using natural occurring standard Calichemicin variants α’3 and γ’1. The library
involved the exchange of natural 3’O methlyrhamnose (red) in the Calcheamicin variants
with sugars supplied via the 10 established CalG1 NDP-sugar substrates (Figure 9).
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Once the library was constructed, the products of these reactions were analyzed
using LC-MS/MS and the resulting fragmentation data was similar to the in vitro reaction
of Figure 7A. The similarity in fragmentation results of the in vitro study in comparison
with naturally occurring standard Calcheamicin variants showed attachment of a sugar to
the aromatic ring of the substrate which further designated CalG1 as the Calcheamicin
GT that was flexible towards diverse TDP-D- and TDP-L- sugar donors which leads to
the synthesis of exotic sugars.
The exchange of native natural-product glycosides with exogenous carbohydrates
supplied as NDP-sugars
To further verify the regiospecificity of CalG1, Calicheamicin
I
3 (Compound
2
from figure 7B) and TDP-3-deoxy- -D-glucose were incubated in a similar reaction to
that of figure 7A. Calicheamicin
I
3 has
a CalG1 glycosylation site occupied by 3’-O-
methylrhamnose so therefore, no product was expected. However, two products were
formed which was identified by LC-MS/MS as aglycon 1 at a peak of 16 minutes, and
the corresponding 3-deoxyglucoside, 2c at about 14.5 minutes (Figure 7D, iv and v).
This reaction was compared to the control reaction of panel ii (Figure 7D) and the
consensus was that a transformation occurred that involved a TDP dependent reverse
glycosyl transfer. The products of the reverse reaction were also analyzed using anionexchange high performance liquid chromatography (HPLC) (Figure 7C) with panel i
representing the control and panel ii being the glycosyl transfer reaction. The new peak
at 13 min was isolated and identified as TDP-3-O-methyl-β-L-rhamnose 3. This analysis
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showed an abundant production of TDP-3-O-methyl-β-L-rhamnose 3 which was
unavailable in the control assay.
What had occurred in this reaction was that in the presence of TDP, CalG1
excised the native Calcheamicin 3’-O methlyrhamnosyl unit, which resulted in the
production of aglycon 1 and TDP sugar 3 (Figure 7A). Also, when a slight excess of
exogenous TDP-3-deoxyglucose was supplied, the GT catalyzed the formation of new
product 2c (Figure 7D, panel iv). The newly discovered in situ “sugar exchange” offers
to researchers a way to substitute CLM 3'-O-methylrhamnose with other natural or
unnatural sugars4.
In order to test this hypothesis, several Calcheamicin derivatives were assayed
(Figure 8) in CalG1-catalyzed reactions with the 10 established CalG1 TDP-sugar
substrates (Figure 9). Every reaction with a Calcheamicin derivative yielded the desired
sugar exchange which was analyzed with HPLC with an average sugar exchange of 60%
for the eight Calcheamicin aglycons in the presence of purified TDP- -D-glucose or
TDP-ß-L-rhamnose 4. With the development of this simple assay, the scientists were able
to produce a Calcheamicin library containing about 70 variants (Figure 10).
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Figure 7. In vitro CalG1-catalyzed reactions. (A) The CalG1-catalyzed transfer of
unnatural sugars to the acceptor 1 yielding a new product. (B) Reaction of
glycosyltransfer. (C) Anion exchange HPLC of CalG1 catalyzed formation. (D) Reversephase HPLC of CalG1 catalyzed.4
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Figure 8. The Construction of the Calicheamicin Library.4
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Figure 9. The structures of the TDP sugars tested in the making of the Calicheamcin
Library. 4
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Figure 10 Efficiency of CalG1-catalyzed ’sugar exchange’ reactions.
Figure 10 illustrates the efficiency of CalG1-catalyzed “sugar Exchange”
reactions which was carried out by the incubation of Calcheamicin (2, 4-9) with the TDP
sugars provided (Figure 9) in the presence of CalG1. The reactions were then analyzed by
means of RP-HPLC. The percent conversions for the sugar products were calculated from
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the corresponding HPLC traces by dividing the integrated area of glyosylated products by
the sum of the integrated are of the product and remaining Calchemicin substrates. 4
The transfer of a sugar from one natural product backbone to a distinct naturalproduct scaffold.
The results of the above reactions proved that for a GT catalyzed sugar exchange
to occur, an established NDP-sugar intermediate needed to be present. From this, the
scientists hypothesized that in a single reaction, a GT could be used to harvest an exotic
sugar from one natural product scaffold and transfer it to a different aglycon.4 Being able
to do this would allow scientists to avoid the often complex synthesis of highly
functionalized NDP sugars.
This reaction of the transfer of exotic sugars to different aglycons involved assays
that contained CalG1, a putative 3’-O-Methylrhamnose donor (Figure 8), TDP, and the
representative acceptor 1. Each assay resulted in simultaneous excision and in situ
transfer of 3’-O-methyrhamnose from each donor to 1, which yielded the expected 3’-Omethlyrhamnosylated product 2. (Figure 11). These results were compared to control
assays that lacked CalG1, or TDP and the reaction only gave starting materials. Being
able to accomplish an in situ aglycon exchange reaction allows for a wider potential of
diversity in accessing CalG1. 4
16
Figure 11. A representative of a CalG1-catalyzed aglycon exchange reaction. (A)
Scheme for a representative CalG1-catalyzed aglycon exchange. (B) RP-HPLC analysis
of CalG1-mediated transformations 4.
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The Testing of reversibility on other GT systems
This study was able to establish CalG1 as the GT capable of reverse catalyzed
sugar exchange and aglycon exchange transformations. This study also raised the
question as to whether the same results would occur in the presence of other GT systems.
To answer this question, another GT catalyzed reaction was examined containing a
different GT known as CalG4 (the putative Calcheamicin aninopentosyltransferase).
CalG4 was produced in a similar fashion as that of CalG1. In the same manner as
CalG1, CalG4 catalyzed the excision of the aminopentose sugar moiety from the sugar
donor Calcheamicin derivatives-4, 5, 6, and 8 (Figure 12). CalG4 was also able to
catalyze the in-situ aglycon exchange, transferring the excised aminopentoses from
donors 4,5,6,7 and 8 to the exogenous aglycon acceptor 1 in the presence of TDP.
(Figure 13). This reaction was compared to controls lacking TDP or CalG4 and again,
only the starting materials were produced. This experiment was also able to establish
CalG4 as the aminopentosyltransferase involved in Calcheamicin biosynthesis, and it also
confirmed that, in contrast to the previously proposed UDP sugar pathways, 10
Calcheamicin aminopentose biosynthesis proceeds via a TDP-sugar pathway. Above all,
this study proved that the reversibility of GTs is not only unique to CalG1.
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Figure 12. CalG4-catalyzed reverse glycosyltransfer. (A) Scheme for CalG4-catalyzed
reverse reactions. (B) RP-HPLC analysis of TDP-dependent reverse CalG4 catalysis. 4
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Figure 13.CalG4-catalyzed aglycon exchange reactions. (A) Scheme for CalG4catalyzed aglycon exchange reactions. (B) RP-HPLC analysis of CalG4-catalyzed
aglycon exchange reactions. 4
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The exploitation of GT-catalyzed reaction reversibility provides an additional use
for glycosylation as a tool to change and vary the activity of therapeutically important
natural products 5. For example, before this work, only two methods for differentially
glycosylating Calcheamicins were available: pathway engineering and total synthesis.
Pathway engineering is a very powerful derivatization tool used for certain natural
products 11 however, it usually turns out badly when used on Calcheamicin-producing M.
echinospora and because of this, many of scientists find this method very impractical
and very cost inefficient. 11. With the newly found reversibility of GTs, it is possible to
exploit these reactions so as to synthesize rare and necessary NDP-sugars, exchange one
sugar scaffold from one core to the other, and also, it versatility will allow for synthesis
of variants of anticancer drugs or drugs in general that are needed for the well being of
others. With the reversibility of GTs, the problem of shortages of drugs such as
Calcheamicin can be avoided because equally as good and effective variants can be
created to address the problem.
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References
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4. Zhang, C., Griffith, B., Quiang, F., Albermann, C., Fu, X., Lee, I., Lingjun, L.,
Thorson, J. Science 2006 313, 1291-1294.
5. Claiborne, C.F; Theodorakis, E.A; Nicolaou, K.C. Pure &Appl. Chem (1996) 68,
2129-2136.
6. J. Ahlert et al., Science 2002, 297, 1173-1173.
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9. X. Fu Nat. Biotechnology. 2003, 21, 1467
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2129-2136.
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