Molecular basis for trehalase inhibition revealed by the structure of trehalase in complex with potent inhibitors

Article abstract of DOI:10.1002/anie.200604825

Strong inhibitions: The inhibition of trehalases, enzymes which hydrolyze the disaccharide trehalose, is a target for novel antibiotic insecticides. The structures (see picture; C black, N blue, O red, S yellow) of a trehalase in complex with validoxylami

Full text of DOI:10.1002/anie.200604825

Angewandte
Chemie
DOI: 10.1002/anie.200604825
Enzyme Structures
Molecular Basis for Trehalase Inhibition Revealed by the Structure of
Trehalase in Complex with Potent Inhibitors**
Robert P. Gibson, Tracey M. Gloster, Shirley Roberts, R. Anthony J. Warren,
Isabel Storch de Gracia, ngela García, Jose L. Chiara, and Gideon J. Davies*
Studies of the unusual nonreducing disaccharide trehalose (a-
d-glucopyranosyl a-d-glucopyranoside; 1; Scheme 1) and its
demonstrated that trehalose plays a vital role as a signaling
molecule in plant development.
[
3]
Trehalose is particularly important for insects, as it is
hydrolyzed into glucose, which is vital for insect flight. The
inhibition of the breakdown of trehalose is, thus, an interest-
ing target for novel insecticides. The hydrolysis of trehalose is
catalyzed by glycoside hydrolases, trehalases, which fall into
family GH37 of the Carbohydrate-Active Enzyme (CAZy)
[
4]
classification. Although approximately 130 open reading
frames are classified in this family, no three-dimensional
structure was available for any of these enzymes prior to this
work. GH37 trehalases (Enzyme Commission (EC) number
3
.2.1.28) hydrolyze one of the two glycosidic bonds in
[
5]
trehalose with inversion of the anomeric configuration.
[
6,7]
Whilst many trehalase inhibitors are active as insecticides
for example, 2 and 3; Scheme 1), the details of their mode of
(
Scheme 1. Trehalose (1), and the trehalase inhibitors validoxylamine A
action were previously unknown. Here we present the first
three-dimensional structure of a trehalase, the periplasmic
Tre37A enzyme from Escherichia coli, which was solved by
single-wavelength anomalous dispersion (SAD) methods
with diffraction data to 1.5-Š resolution. The enzyme
structure was determined in complex with the potent
inhibitors validoxylamine A (2) and 1-thiatrehazolin (4;
Scheme 1). Compounds 2–4 have been shown to have kinetic
inhibition (K) and calorimetric dissociation (K ) constants in
the low nanomolar range, with the interactions being
characterized by a large favorable enthalpy change (DH).
Tre37A was prepared by periplasmic expression using its
own signal peptide. This protein was used for all subsequent
kinetic, calorimetric, and structural studies. Enzymatic activ-
ity was measured using an assay in which glucose was detected
using glucose oxidase/peroxidase linking enzymes, following
trehalose hydrolysis. At pH 5.5 and 378C, Tre37A has a
(2), trehazolin (3), and 1-thiatrehazolin (4). The numbering of 2–4 was
chosen to aid comparison with trehalose.
metabolism date back to the isolation of trehalose from the
[
1]
“
trehala” cocoons of weevils of the genus Larinus. A variety
of physiological roles for the disaccharide have been demon-
strated in organisms ranging from bacteria and fungi to
invertebrates and higher plants. Trehalose is a common source
of energy, and its role as a universal stress protectant is also
becoming increasingly clear. Cellular damage resulting from
temperature extremes, osmotic/oxidative stress, toxic chem-
ical exposure, desiccation, hydrostatic-pressure changes, or
nutrient starvation has an effect on the production and
i
d
[
2]
utilization of trehalose. Other studies have revealed that
trehalose forms the backbone of the structural cord factor
from Mycobacterium tuberculosis, and recent work has
Michaelis constant (K ) of 0.41 mm and a turnover number
M
À1
(
kcat) of 199 s (Figure 1). Compounds 2–4 have all previously
[*] Dr. R. P. Gibson, Dr. T. M. Gloster, S. Roberts, Prof. R. A. J. Warren,
been reported to be tight-binding slow-onset inhibitors of
Prof. G. J. Davies
Department of Chemistry
[
8,9]
trehalase,
notably with the pig-kidney enzyme. In our
laboratory, trehazolin (3) decayed after storage at À208C,
highlighting the advantages of 1-thiatrehazolin (4), which
remains stable for (at least) several years. Inhibition studies
York Structural BiologyLaboratory
York, YO105YW (UK)
Fax: (+44)1904-328266
E-mail: davies@ysbl.york.ac.uk
(performed following a 20-min pre-incubation between the
I. Storch de Gracia, . García, Dr. J. L. Chiara
Instituto de Química Orgµnica General, CSIC
Juan de la Cierva, 3
enzyme and inhibitor to mitigate slow-onset inhibition) show
that 2 and 4 are indeed very potent inhibitors of Tre37A, with
K values of 10 and 9 nm, respectively. Isothermal titration
i
28006 Madrid (Spain)
calorimetry (carried out at pH 7 and 258C) also revealed that
both 2 and 4 are tight-binding inhibitors of Tre37A, with Kd
values of 7 and 15 nm, respectively (Figure 2). Both binding
events are characterized by a large favorable enthalpy of
[
**] R.P.G. and T.M.G. contributed equallyto this work. This work was
funded bythe Biotechnologyand Biological Sciences Research
Council (BBSRC). G.J.D. holds a Royal Society Wolfson Merit Award.
The authors thank AnthonyO’Sullivan (S yn genta) for the provision
of compound 2 and for useful discussions.
À1
binding (DH values of À18.9 and À12.7 kcalmol for 2 and 4,
Angew. Chem. Int. Ed. 2007, 46, 4115 –4119
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Communications
Figure 1. Michaelis–Menten kinetics for the hydrolysis of trehalose by
Tre37A; the reaction rate is plotted against trehalose concentration.
respectively), but an unfavorable entropy change (TDS
À1
values of À7.8 and À2.1 kcalmol for 2 and 4, respectively).
The three-dimensional structure of Tre37A was solved
using SAD methods with a selenomethionine (SeMet)
derivative of the enzyme, which was initially crystallized in
the presence of 2. X-ray diffraction data were collected to 2.2-
Figure 2. Isothermal titration calorimetry(ITC) data for the binding of
to Tre37A. Top: Raw titration data of the power supplied to the
Š
resolution, and the Tre37A model could be traced from
4
Pro37 to Pro547 (final Rcryst of 0.16 and Rfree of 0.20), with
some of the surface residues being disordered. Subsequently,
the protein coordinates from this structure were used as the
starting model for the refinement of the structure of a Tre37A
complex with 4, with data to 1.5-Š resolution (final Rcryst of
system to maintain a constant temperature plotted against time.
Bottom: The bimolecular fit of the normalized heats of interaction
plotted against the molar ratio of 4 to Tre37A.
0
.15 and Rfree of 0.19).
The structure of Tre37A, the first of a trehalase, consists of
The complexes of Tre37A with 2 and 4 (Figure 3b,c)
reveal the interactions in the À1 and + 1 subsites. In both
complexes, the positions of the protein side chains are
invariant, but intriguingly, the disaccharide mimics are
completely buried within the structure. Significant conforma-
tional change would be required for substrate entry and
departure into the occluded active centre. Such conforma-
tional change may contribute both to the slow onset of
an (a/a) barrel (Figure 3a) similar to that found for other a-
toroidal glycosidases, notably GH94 chitobiose phosphory-
lases,
phorylases. All of these enzymes display the (a/a) toroid
fold; they are classified in the glucoamylase family by the
secondary-structure matching (SSM) server, and all per-
form catalysis with inversion of the anomeric configuration, as
is the case for the GH37 trehalases.
6
[
10]
[11]
GH15 glucoamylases,
and GH65 maltose phos-
[
12]
6
[
13]
[
9]
inhibition observed in kinetic studies by others and the
unfavorable entropy of interaction. In the leaving-group + 1
Figure 3. a) Ribbon representation of Tre37A in complex with validoxylamine A (2), color-ramped from the N terminus (blue) to the C termi-
nus (red); 2 and the residues proposed to act as the acid (Asp312) and base (Glu496) in the hydrolysis mechanism are shown in ball-and-stick
representation. Ball-and-stick representations of b) 2 and c) 1-thiatrehazolin (4) bound to Tre37A (residues Glu496 and Asp312 are shown; +1
and À1 subsites are labeled); the observed electron densityfor the maximum-likelihood weighted 2 FobsÀFcalcd map is contoured at 1s. C black,
[
14]
N blue, O red, S yellow. The figure was drawn using BOBSCRIPT.
4
116
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Angewandte
Chemie
subsite, the pseudosugar ring of 2 and the glucose moiety of 4
leaving-group departure is complemented by Brønsted base
4
(
which both have a C chair conformation) lie in a nearly
assistance to nucleophilic attack by a water molecule, in a
single-displacement mechanism. The structure does not sup-
port a mechanism involving ring opening, which was once
1
identical position (Figure 4), which promotes favorable
hydrogen bonding with residues Arg152, Asn196, Arg205,
Arg277, Glu279, and Glu511 (Figure 5). As was also found for
[
5]
considered. The complexes of Tre37A with 2 and 4, and the
similarity of the active centre to those of glucoamylases,
strongly implicate Asp312 (spatially approximate to Glu179
[
11]
of Aspergillus glucoamylase ) and Glu496 (directly equiv-
[
11]
alent to Glu400 of Aspergillus glucoamylase ) as the
catalytic acid and base, respectively, in the inversion mech-
anism. Asp312 is poised 2.8 Š from the “glycosidic” nitrogen
atom of both 2 and 4 (Figures 3 and 4). Glu496 sits above the
b face of the pseudosugar ring in 2, and is poised to activate a
water molecule (located 4 Š from C1 of 2) for nucleophilic
attack. In the complex with 4, no electron density that could
be assigned to a potential water molecule is detected, as the
hydroxy group at C5 of 4 occupies a similar position to that
occupied by the putative attacking water molecule in the
complex with 2. The hydroxy group of Tyr512 may impede the
attacking water molecule, which implies that small conforma-
tional rearrangements of the determined structures are
necessary for catalysis.
Figure 4. Ball-and-stick representation of the superposition of validox-
ylamine A (2; yellow) and 1-thiatrehazolin (4; blue) bound to Tre37A
(
residue Asp312 is shown; +1 and À1 subsites are labeled). C black,
N blue, O red, S yellow.
In the À1 subsite, there are differences in the positions of
the valienamine and tetrahydrocyclopentathiazole moieties
of 2 and 4, and the interactions they make with Tre37A. In the
2
other enzymes such as glucoamylases, there is no classical
hydrophobic platform in this subsite. This feature, in tandem
with the hydrogen-bonding template, provides the driving
force for the accommodation of the a,a-1,1-linked disacchar-
ide, as opposed to a-1,4-linked substrates. The extensive
hydrogen bonding and the lack of hydrophobic stacking in the
“catalytic” À1 subsite, the valienamine ring of 2 has a H half-
3
chair conformation, as dictated by the double bond between
C5 and C6, whilst the cyclopentane ring of 4 adopts a E
envelope conformation. As expected, 2 binds essentially as
seen previously for the inhibition of a-1,4-glucan-hydrolyzing
2
[
11,17]
enzymes by acarbose.
Surprisingly, despite substantial
+
1 subsite provide a persuasive structural rationale for the
differences in their chemical structures, 2 and 4 make similar
interactions with the protein.
reduced affinity of simplified hydrophobic derivatives of 2–
[
15,16]
4.
The hydroxy groups at C3 and C4 of 2 superimpose well
with their equivalents in 4 (Figure 4); in both cases, these
hydroxy groups make hydrogen bonds with Gly310 (main
chain), Trp159, Gln207, and Asp160. The hydroxy group at C2
of 2 interacts with Trp447 and Asp312, and although 4
possesses no equivalent exocyclic substituent, the sulfur atom
of the thiazoline ring makes comparable interac-
tions. In a similar compensatory way, the hydroxy
group of the hydroxymethyl group at C5 of 2
hydrogen bonds with Asp160, as does the axial
hydroxy group at C5 of 4 (Figures 4 and 5). In both 2
and 4, the nitrogen atom equivalent to the glycosidic
oxygen atom of trehalose interacts with the catalytic
acid Asp312. Presumably, the favorable geometry of
this interaction, enhanced by the potentially tran-
sition-state-mimicking conformations of the two
moieties in the À1 subsite, contributes significantly
to the high affinity. Furthermore, the nitrogen atom
of the thiazoline ring of 4 lies in the position that the
endocyclic O5 atom would occupy in the natural
substrate, which is likely to contribute to the
favorable enthalpy of interaction of 4.
Catalysis by trehalases is known to occur with inversion of
the anomeric configuration, as is also the case for its closest
structural homologues from families GH15 and GH65.
Glycoside hydrolysis with inversion of the anomeric config-
uration follows a canonical mechanism: proton assistance to
A number of other glycosidase inhibitors feature
thiazoline moieties. Allosamidin, for example, pos-
sesses a tetrahydrocyclopentoxazole ring system
Figure 5. Interactions between 1-thiatrehazolin (4) and Tre37A. Hydrogen bonds
are shown bydashed lines.
similar to that of 4 and is a potent chitinase
Angew. Chem. Int. Ed. 2007, 46, 4115 –4119
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4117

Communications
[
18]
[27]
inhibitor. Similarly, thiazoline derivatives of N-acetylglu-
atom phasing was performed with autoSHARP, subsequent model
[
28]
[29]
[
19]
building with COOT,
and refinement with REFMAC.
atomic coordinates have been deposited in the Protein Data Bank
PDB) with accession codes 2JF4 (SeMet Tre37A with 2) and 2JG0
Tre37Awith 4); additional experimental details are given in the PDB
The
cosamine are tight-binding hexosaminidase inhibitors. In
this case, however, the thiazoline moiety mimics the covalent
oxazolinium ion intermediate, or a closely related transition
(
(
[
20]
state, formed during the substrate-assisted double-displace-
ment reaction of a retaining glycosidase. In contrast, 3 and 4
inhibit an inverting glycosidase, and their favorable tight
binding reflects an adventitious mimicry of the hydrogen
bonding of the transition state during a single-displacement
reaction mechanism. This situation is similar to general
glycosidase inhibition using five-membered rings (recently
headers.
The kinetics of the hydrolysis of trehalose by Tre37A was
investigated using a stopped assay, in which glucose was detected
using glucose oxidase/peroxidase linking enzymes (Megazyme).
Assays were performed at 378C in sodium maleate buffer (75 mm,
À1
pH 5.5) containing bovine serum albumin (BSA; 0.38 mgmL ).
Measurements were made at trehalose concentrations of 0.05–4 mm;
Tre37Awas present at a final concentration of 5.4 nm. 200-mL aliquots
of the reaction mixture were taken at time intervals, and after boiling
the aliquots for 2 min, 1 mL of the glucose oxidase/peroxidase
solution was added. Assays were otherwise performed as described
in the manufacturerꢀs instructions. The reaction rates were deter-
mined and the data were fitted to the Michaelis–Menten equation
using GRAFIT (Erithacus Software Ltd.). The inhibition constant
[
21]
described for mannostatins ). The exact nature of the
transition state conformation for glycoside hydrolysis by
4
trehalases remains undefined, with both H half-chair and
3
2
,5
B boat conformations of the six-membered rings being
[
22,23]
possible (by virtue of work on analogous systems
). A
feature of compounds 2–4 is that, whilst none closely mimic
(
K) values were determined in the same way, in the presence of 2 or 4
4
2,5
i
either the H or the B conformation, all are reasonable
3
(10 nm) and at a final Tre37A concentration of 1 nm (to ensure a 10-
mimics of both conformations. To some extent, the conforma-
tional flexibility of the hydroxymethyl group at C5 of 2 and of
both the axial and the equatorial substituents at C5 in 3 and 4,
allows the compounds to optimize hydrogen bonding, despite
the apparent differences in ring conformations. Such strat-
egies to overcome the chemical challenges of transition-state
mimicry, which build on the benefits of aglycon interactions
fold excess of the inhibitor). Tre37A was pre-incubated with each of
the inhibitors for 20 min prior to the start of the assay to prevent any
complications with slow-onset inhibition. The data were similarly
fitted using GRAFIT to obtain an apparent Michaelis constant
app
app
(
KM ), and K values were determined using the equation K
=
i
M
K (1+[I]/K), where [I]is the concentration of the inhibitor.
M
i
Isothermal titration calorimetry (ITC) was performed using a VC
calorimeter (Microcal) at 258C. Tre37A (31–37 mm) was dialyzed into
sodium phosphate buffer (50 mm, pH 7.0), and inhibitors 2 and 4
(0.5 mm) were diluted in the same buffer. Titrations were performed
by injecting 10-mL aliquots of the ligand into Tre37A. The data were
corrected for the heat of dilution by subtracting the heat at a high
molar ratio of inhibitor to enzyme. The stoichiometry (n), enthalpy
(
as illustrated by compounds 2–4), should provide inspiration
for future generations of glycosidase inhibitors.
Experimental Section
change (DH), and equilibrium association constant (K ) were
a
The gene encoding Tre37A was amplified by the polymerase chain
reaction (PCR) and ligated into the NdeI and XhoI sites of a pET22b
expression vector (Novagen) for periplasmic expression with the
native Tre37A signal peptide. E. coli BL21 (DE3) cells were
transformed with the plasmid containing the Tre37A gene and
determined by fitting a bimolecular model to the data using Microcal
Origin. The Gibbs free energy (DG) and TDS were calculated using
the equations DG = ÀRTlnK = DHÀTDS.
a
Received: November 28, 2006
Published online: April 23, 2007
grown at 378C in Luria–Bertani (LB) broth containing ampicillin
À1
(
50 mgmL ), until an optical density at 600 nm (OD600) of approx-
imately 0.7 AU was reached. Over-expression was initiated by the
addition of isopropyl thio-b-d-galactoside to a final concentration of
Keywords: inhibitors · protein structures · trehalases ·
trehazolins · validoxylamines
.
1
mm, and cells continued to grow at 308C for 12 h. Cells were
harvested by centrifugation, and Tre37A was obtained by osmotically
shocking the cells. Tre37A was purified by ion-exchange chromatog-
raphy (HiTrap MonoQ, Amersham Biosciences), and by gel filtration
(
Superdex 200 16/60), using standard procedures. A selenomethio-
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[
24]
induction methodology of Studier and purified in the same way.
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118
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ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2007, 46, 4115 –4119

Angewandte
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Angew. Chem. Int. Ed. 2007, 46, 4115 –4119
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
4119