Structural characterization of O- and C-glycosylating variants of the landomycin glycosyltransferase LanGT2

Article abstract of DOI:10.1002/anie.201409792

The structures of the O-glycosyltransferase LanGT2 and the engineered, C-C bond-forming variant LanGT2S8Ac show how the replacement of a single loop can change the functionality of the enzyme. Crystal structures of the enzymes in complex with a nonhydroly

Full text of DOI:10.1002/anie.201409792

Angewandte
Chemie
DOI: 10.1002/anie.201409792
Antibiotics
Structural Characterization of O- and C-Glycosylating Variants of the
Landomycin Glycosyltransferase LanGT2**
Heng Keat Tam, Johannes Hꢀrle, Stefan Gerhardt, Jꢁrgen Rohr, Guojun Wang, Jon S. Thorson,
Aurꢂlien Bigot, Monika Lutterbeck, Wolfgang Seiche, Bernhard Breit,* Andreas Bechthold,* and
Oliver Einsle*
Abstract: The structures of the O-glycosyltransferase LanGT2
sity of a range of therapeutically important natural products,
they have largely been restricted to O-glycosylation and, to
a lesser extent, N-glycosylation.[4] The inclusion of C-glyco-
À
and the engineered, C C bond-forming variant LanGT2S8Ac
show how the replacement of a single loop can change the
functionality of the enzyme. Crystal structures of the enzymes
in complex with a nonhydrolyzable nucleotide-sugar analogue
revealed that there is a conformational transition to create the
binding sites for the aglycon substrate. This induced-fit
transition was explored by molecular docking experiments
with various aglycon substrates.
À
syltransferases, which are able to form C C bonds, into this
scheme would thus pave the way for the production of novel
bioengineered metabolites.[5]
LanGT2 is an O-glycosyltransferase (O-GT) that cata-
lyzes the initial glycosylation of the aglycon in the biosynthe-
sis of landomycin A, and it shares high homology with the C-
glycosyltransferase (C-GT) UrdGT2 from urdamycin A bio-
synthesis. Both enzymes transfer d-olivose from NDP-d-
olivose to closely related angucycline-based aglycons to yield
the respective O- or C-glycoside products (Figure 1). LanGT2
catalyzes the transfer of d-olivose to an aglycon C8-OH,
G
lycosylated natural products predominate in antimicrobial
and anticancer drug discovery. Often, the biological activity
and pharmacological properties of the compounds are
determined by attached sugar moieties[1] that also form part
of the cellular defense strategy of antibiotic producers.[2] The
stereo- and regiospecific glycosylation catalyzed by glycosyl-
transferases (GT) typically occurs during biosynthesis, as
a multistep modification of complex core aglycons that may
be secondary metabolites such as polyketides. The permissive
nature of glycosyltransferases has allowed the differential O-
glycosylation of a range of complex natural products and
drugs through in vitro and in vivo strategies.[3] While such
approaches have dramatically extended the structural diver-
[*] Dr. H. K. Tam, Dr. S. Gerhardt, Prof. Dr. O. Einsle
Institut fꢀr Biochemie, Albert-Ludwigs-Universitꢁt Freiburg
Albertstrasse 21, 79104 Freiburg (Germany)
E-mail: einsle@biochemie.uni-freiburg.de
Dr. J. Hꢁrle, Prof. Dr. A. Bechthold
Institut fꢀr Pharmazeutische Wissenschaften
Albert-Ludwigs-Universitꢁt Freiburg, 79104 Freiburg (Germany)
E-mail: andreas.bechthold@pharmazie.uni-freiburg.de
Figure 1. Structures of the angucycline antibiotics landomycin A and
urdamycin A. The O-GT LanGT2 attaches d-olivose to the aglycon
through an O-glycosidic bond, while the C-GT UrdGT2 links the same
À
carbohydrate moiety through a C C bond to the aglycon of urdamy-
cin A.
Prof. Dr. J. Rohr, G. Wang, Prof. Dr. J. S. Thorson
Center for Pharmaceutical Research and Innovation
University of Kentucky College of Pharmacy, Lexington, KY (USA)
Dr. A. Bigot, M. Lutterbeck, Dr. W. Seiche, Prof. Dr. B. Breit
Institut fꢀr Organische Chemie, Albert-Ludwigs-Universitꢁt Freiburg
Albertstrasse 21, 79104 Freiburg (Germany)
while UrdGT2 attaches the same sugar directly to C9 of the
aglycon, with inversion of the anomeric stereocenter in both
cases. The aglycon substrate for UrdGT2 is 2-hydro-3-
hydroxy-prejadomycin (UWM6),[6] whereas the exact phys-
iological aglycon substrate for LanGT2 is not known.
Expression of urdGT2 in a lanGT2-deficient strain of S.
cyanogenus S136 resulted in the formation of 9-C-olivosylte-
trangulol, while the expression of lanGT2 in the same mutant
strain yielded 8-O-olivosyl-d-11-deoxylandomycinone.[7]
LanGT2 and UrdGT2 share 53% sequence identity and
were presumed to be similar in structure and mechanism, thus
inspiring attempts to modify their substrate specificities by
E-mail: bernhard.breit@chemie.uni-freiburg.de
Prof. Dr. O. Einsle
BIOSS Centre for Biological Signalling Studies
Schꢁnzlestrasse 18, 79104 Freiburg (Germany)
[**] This study was supported in part by Deutsche Forschungsgemein-
schaft (GSC-4, Spemann Graduate School of Biology and Medicine,
SGBM), NIH (GM 105977-01A1 to J.R. and R37 AI52218 to J.S.T.),
and by an EMBO Short Term Fellowship (ASTF 261-2012) to H.K.T.
We thank the staff at the Swiss Light Source, Villigen, Switzerland,
for assistance during data collection.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201409792.
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swapping relevant amino acid motifs between the enzymes.[8]
A prime goal was to generate C-glycosylating variants of
LanGT2, and this goal was eventually achieved by grafting
residues 51VATTDLPIRHFI62 of UrdGT2 into LanGT2.[7b]
Furthermore, an S8A variant of the chimeric LanGT2
(LanGT2S8Ac), in which a signature residue (glycine or
alanine) of the C-GTs UrdGT2, SimB7, HedL, and SsfS6[9]
was introduced, further increased the C-glycosylation effi-
ciency.[7b] Similarly, a recent mutagenesis study focusing on
a Pyrus communis O-GT (PcOGT) and an Oryza sativa C-
GT (OsCGT) revealed a single mutation of PcOGT, namely
D118I, that led to engineered C-GT activity.[10]
trizing directed hydroformylation developed recently in our
laboratory, followed by an intramolecular carbonyl–ene
reaction.[11] After reductive removal of the chiral o-DPPF
directing group and ozonolysis, a directed carbonyl reduction
delivered the carbaolivose backbone with good overall yield
and stereoselectivity. Standard functional group manipula-
tions enabled the introduction of the thymidine diphosphate
moiety to furnish (À)-8 in enantiomerically pure form (see the
Supporting Information).
The molecular features that distinguish O- and C-specific
GTs remain poorly understood. Herein, we report the three-
dimensional structures of LanGT2 and LanGTS8Ac in
complex with the surrogate sugar nucleotide ligands TDP-
carba-d-olivose and thymidine diphosphate. Our data suggest
that both O- and C-GTs utilize conserved amino acid residues
for general acid–base catalysis. The key feature that differ-
entiates O-glycosylation from C-glycosylation is the specific
orientation of the sugar nucleotide with respect to the
nucleophile. The structural consequence of the mutations in
LanGT2S8Ac is a reorientation of the aglycon to favor C-
glycosylation. Our study helps to rationalize the altered
specificity of the chimeric enzyme and provides the first
structural template for understanding engineered C-GT
activity.
Both LanGT2 and the engineered LanGTS8Ac were
crystallized and their structures were determined by X-ray
diffraction. They show the typical two-domain architecture of
their class, with an N-terminal aglycon-binding domain and
a C-terminal nucleotide-sugar-binding domain (Figure 2A).
The structure of LanGT2S8Ac was highly similar to that of
the wild type, with the major difference occurring in the
grafted loop from residues 51–62 (Figure 2B). This region is
structurally distinct in LanGT2 and UrdGT2,[8a] but within the
LanGT2 background of the chimera, it bears a striking
resemblance to the original UrdGT2 structure (Figure 2C).[8a]
The conservation of the loop structure in this engineered C-
glycosylating enzyme thus supports the hypothesis that the
positioning of the aglycon is a primary determinant of the
resulting mode of glycosylation.
Figure 2. Three-dimensional structures of LanGT2. A) The LanGT2
monomer colored from blue at the N terminus to red at the C termi-
nus. The protein is organized as a nucleotide-sugar-binding domain
(top) and an aglycon-binding domain (bottom) that are connected
through a flexible hinge. B) A 908 rotation highlights the grafted loop
at the rim of the N-terminal domain. C) Detail of the grafted region in
LanGT2 (red), LanGT2S8Ac (blue), and UrdGT2 (purple, PDB-ID
2P6P). The UrdGT2-derived region retains its conformation in the
LanGTS8Ac chimera. The box in (B) highlights this region in LanGT2.
The GT reaction follows a sequential bi–bi mechanism,
wherein the nucleotide sugar is bound first, followed by
binding of the aglycon and sugar transfer. We therefore tried
to generate structures for LanGT2 and LanGT2S8Ac in
complex with TDP-olivose in order to prepare the ground for
aglycon binding studies. Unfortunately, the TDP-olivose
complex proved to be unstable on the timescale of crystal-
lization, thus resulting in structures that invariably only had
the product TDP bound (Figure S1 in the Supporting
Information). Therefore, we proceeded to synthesize a non-
hydrolyzable analogue of the activated sugar substrate by
replacing d-olivose by the corresponding carbasugar, carba-
a-d-olivose, based on the assumption that the replacement of
the ring oxygen by carbon atom would prevent glycosyl
transfer (Scheme 1).
LanGT2 crystals were soaked with the synthetic TDP-
carba-d-olivose (TcO) and LanGTS8Ac was cocrystallized
with TcO according to the same protocols that merely yielded
the TDP-bound structures when TDP-olivose was used. The
structures of the two complexes were solved to 1.85 ꢀ and
2.22 ꢀ resolution, respectively (Table S1 in the Supporting
Information), and they show a binding mode for TDP that is
entirely consistent with the TDP complex and corresponds to
the “tucked-under” conformation of the sugar described for
other GTs.[12] Beside the additional electron density of the
carbasugar moiety, the addition of the ligand also led to
a change in the tertiary structures of the enzymes. The binding
of TDP already induced a rotation of the two domains of the
protein, which resulted in a closure of the interdomain cleft.
With the TcO ligand, however, this closure was more
As a key step for the synthesis of a-d-carbaolivose
thymidine diphosphate (8), we applied a one-pot desymme-
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similar cytosine. This
arrangement is con-
served among GT-1
family
members,
including the represen-
tative CalG1, CalG2,
and SpnG.[14] On the
side of the base distal
from W267, the side
chain of L270 is situ-
ated above the base, in
a motif that is typically
observed in TDP-
sugar-utilizing
GTs,
such as GtfA, CalG3,
SpnG, and SsfS6 (Fig-
ure 3B).[14,15] H283 in
LanGT2 and LanGT2-
S8Ac is conserved
throughout the GT-
1 family and has been
proposed to stabilize
the accumulation of
charge on the phos-
phate
during
ca-
talysis.[16] Additionally,
the negative charges of
the central pyrophos-
phate of the ligand are
compensated by posi-
tioning this part of the
molecule at the posi-
Scheme 1. Synthesis of a-d-carbaolivose thymidine diphosphate (8). acac=acetylacetonate, BOP=benzotriazol-1-
yloxytris(dimethylamino)phosphonium, O-isoval=isovalerate, LAH=lithium aluminum hydride, THF=tetrahydro-
furan, TMS=trimethysilyl.
pronounced, thus resulting in a 108 rotation of the C-terminal
with respect to the N-terminal domain (Figure 3A). These
observations indicate an induced-fit mechanism, as has been
described for other GTs.[8b,13] In this “closed state”, additional
secondary structure elements of the protein were defined, in
particular a large helical segment between residues 218 and
228 that was disordered in the absence of a ligand. In
accordance with a sequential binding mechanism for the
activated sugar and the aglycon, we thus conclude that the
actual site for aglycon binding is only formed upon TDP-
olivose binding and is stabilized by entropic effects. During
the transition to the closed state, the loop region S217–F223,
as well as the 283HAGGVT288 motif in the adjacent loop,
reorient to interact with the ligand, while residues S8–A12
and W136–R143 are displaced by carba-d-olivose (Figure S1
in the Supporting Information). Interestingly, helices a8a and
a8b were poorly defined in the TDP-bound structure, thus
indicating that the tightly closed structure of the enzyme had
already relaxed towards an open conformation to release the
product TDP (Figure S2).
tively charged end of helix h10 of the enzyme. In the resulting
binding pocket, multiple residues from the regions 217–220
and 283–288 interact with the two phosphate groups, thereby
providing a stable binding mode that is identical to that
observed for TDP alone (Figure 3B and Figure S3). As in
other glycosyltransferases, recognition of the correct sugar
moiety is achieved through hydrogen bonds. In LanGT2, only
three residues form hydrogen bonds to the sugar, namely
A284, G286 (5’-OH), and D137 (4’-OH; Figure 3B). Note
that D137 is the only residue from the N-terminal aglycon-
binding domain to interact with the activated sugar ligand, so
this domain-spanning interaction may be essential for the
observed conformational change upon ligand binding.
The conformational closure of the enzyme also alters the
shape of the binding pocket, and here notable differences
were observed between LanGT2 and LanGT2S8c. The
resulting aglycon-binding pocket differs in size and shape
between the two enzyme variants and this difference pre-
sumably forms the basis for the different outcome of the
glycosyl transfer reaction.
Two distinct regions of the protein contribute to TDP-
sugar binding. The loop region from W267–D271 mediates
the recognition of the thymine base of TDP, with the indole
side chain of residue W267 forming p-stacking interactions
with the aromatic base, and three hydrogen bonds to the
backbone peptide bonds of residues V268 and L270 providing
the means to distinguish the thymine nucleobase from the
Subsequent soaking and cocrystallization experiments
with the TcO complexes of LanGT2 and LanGT2S8Ac in
combination with various aglycon substrates (11-deoxylando-
mycinone, alizarin, anhydrolandomycinone, or tetrangulol)
did not yield the desired ternary complexes, possibly because
the specificity of the enzymes for the applied aglycons was too
low. We therefore resorted to in silico ligand docking studies
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In the docking models, D137 is
located 3.4 ꢀ away from the C8-OH
group, where it is suggested to recruit
a water molecule to abstract a proton,
thereby substantially increasing the
nucleophilicity of the C8-hydroxy
moiety. For the formation of an O-
glycosidic bond by LanGT2, the C8-
OH is closer to the sugar nucleotide
donor than the ortho C9 atom (Fig-
ure 4A). By contrast, the altered
orientation of the aglycon observed
in the model for LanGT2S8Ac brings
the C9 atom closer to the sugar
nucleotide donor, thus resulting in
À
a C C coupling instead (Figure 4B).
In the models, the distance between
the C8-OH group (11-deoxylandomy-
cinone) or the C9 atom (tetrangulol)
and the C1’ atom of d-olivose is too
large to give the productive orbital
overlap necessary for glycosyl trans-
fer. Catalysis may thus require a fur-
ther rearrangement of the protein,
possibly through the flexible loop
region 64–76, which could act as
a lid to cover the active site cleft.
Herein, we have shown a func-
tional example for the concept of
altering enzymatic specificity by
exchanging specificity-determining
regions between members of the
same protein family. The synthesis
and application of a nonhydrolyzable
carbasugar analogue enabled us to
probe the ordered sequential mecha-
nism of the enzyme and has revealed
an induced-fit mechanism to give the
conformation that binds the aglycon
compound. Grafting a loop region
from UrdGT2 altered the binding
pocket in LanGT2S8Ac so that dock-
ing simulations predict an entirely
different mode of binding that results
Figure 3. Ligand binding to LanGT2. A) Stereo image of a Ca-trace superposition of unbound
LanGT2 (black) and the enzyme with the synthetic analogue TDP-carba-d-olivose (TcO, red)
bound. The ligand induces a 108 rotation of the sugar-binding domain relative to the aglycon-
binding domain (grey arrow). B) Stereo representation of the active site of LanGT2 with bound
TcO. While the loop region 267–271 assures specific binding of the TDP moiety, the negatively
charged phosphodiester is stabilized by the helix dipole of helix h10 (residues 286–295). The 4’ and
5’ hydroxy groups of olivose are recognized by G286 and D137, with the latter being the only
residue from the N-terminal domain that is involved in binding.
based on the available crystal structures and a functional
screening carried out previously.[7b] High-scoring docking
solutions were obtained for 11-deoxylandomycinone and
tetrangulol, where the aglycon substrates interacted with
a series of hydrophobic residues (F83, W87, F88, M91, M116,
and W136) in the interdomain cleft of the glycosyltransferases
(Figure 4 and Figure S4). The aglycon-binding pocket of
LanGT2S8Ac is considerably smaller than that of LanGT2,
an effect that is mainly due to the conformation of helix a3 (in
which residues I58 and I62 are oriented towards the cleft in
the engineered C-GT). D137 is situated adjacent to the C8-
OH group of the aglycon in both models, an observation
consistent with previous studies that support the role of this
residue as a catalytic base.[7b]
in the transformation of the O-glycosyltranferase into a C-
glycosyltransferase.
Received: October 6, 2014
Published online: January 7, 2015
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deoxylandomycinone and LanGT2. In the model, the ligand (shown as van der Waals spheres)
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closing the substrate-binding cleft. Molecular docking suggests that the ligand tetrangulol attains
a different binding mode that reorients R220 towards the substrate and TDP-carbaolivose (TcO),
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Keywords: carbasugars · C-glycosylation · enzyme engineering ·
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