From the Department of Biochemistry and Molecular Biology, The University of Queensland, Brisbane QLD 4072, Australia
Received for publication, November 15, 2002, and in revised form, December 18, 2002
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ABSTRACT |
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Acetohydroxyacid synthase (AHAS) (acetolactate
synthase, EC 4.1.3.18) catalyzes the first step in
branched-chain amino acid biosynthesis and is the target for
sulfonylurea and imidazolinone herbicides. These compounds are
potent and selective inhibitors, but their binding site on AHAS has not
been elucidated. Here we report the 2.8 Å resolution crystal structure
of yeast AHAS in complex with a sulfonylurea herbicide, chlorimuron
ethyl. The inhibitor, which has a Ki of 3.3 nM, blocks access to the active site and contacts multiple
residues where mutation results in herbicide resistance. The
structure provides a starting point for the rational design of further
herbicidal compounds.
Herbicides are widely used for weed control in agriculture and
industry and are also used by government agencies and home gardeners.
It is estimated that worldwide sales of herbicides exceed $30 billion,
with the sulfonylureas (Fig.
1a) and imidazolinones (Fig.
1b) accounting for about $2 billion in annual sales. The sulfonylureas and imidazolinones act by preventing branched-chain amino
acid biosynthesis by virtue of their specific and potent inhibition of
acetohydroxyacid synthase
(AHAS)1 (acetolactate
synthase, EC 4.1.3.18), the first enzyme in this pathway (1, 2).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
The chemical structures of: a,
CE, a sulfonylurea herbicide; and b, imazapyr, an
imidazolinone.
AHAS catalyzes the decarboxylation of pyruvate and its combination with another 2-ketoacid to give an acetohydroxyacid (3, 4). The enzyme requires three cofactors: thiamine diphosphate (ThDP), a divalent metal ion such as Mg2+, and FAD. The requirement for the first two of these cofactors is well understood from the chemistry of ThDP and the three-dimensional structure of various enzymes including AHAS (5) and its relatives pyruvate oxidase (6), pyruvate decarboxylase (7, 8), and benzoylformate decarboxylase (9). In contrast, the role of FAD remains puzzling, despite now knowing the location and conformation of this cofactor in the enzyme (5).
The herbicides that inhibit AHAS bear no resemblance to the substrates
and are not competitive inhibitors, suggesting that they bind at a site
distinct from the active site (1, 10-13). Previously, we proposed (5)
a model for the herbicide-binding site, based on the structure of yeast
AHAS and the location of residues where mutation is known to result in
herbicide insensitivity. However, this site is large and exposed to
solvent, and we suggested that structural changes would occur upon
binding of substrates or herbicides. In this paper, we describe the
crystal structure of yeast AHAS in complex with chlorimuron ethyl (CE;
Fig. 1a), a commonly used sulfonylurea herbicide. Our
structure provides the first view of the mode of binding between an
herbicidal inhibitor and AHAS and elucidates the location of the
herbicide resistance mutations in this enzyme.
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EXPERIMENTAL PROCEDURES |
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Expression, Purification, Crystallization, and X-ray Data Collection-- The catalytic subunit of yeast AHAS was expressed and purified as described previously (14). Crystals of yeast AHAS were grown by hanging drop vapor diffusion in the presence of 1 mM ThDP, 1 mM MgCl2, 1 mM FAD, 1 mM CE, 5 mM dithiothreitol, 0.2 M potassium phosphate, pH 7.0, 0.1 M Tris-HCl, pH 7.0, 0.2 M Li2SO4, and 0.9 M sodium potassium tartrate. X-ray data (Table I) were collected from cryoprotected crystals (30% v/v ethylene glycol) at 100 K on Beam Line 14D at the Advanced Photon Source in the Argonne National Laboratory (Chicago, IL). The data were indexed, integrated, and scaled using the programs DENZO and SCALEPACK (15).
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Structure Determination-- The crystal structure was solved by molecular replacement using the program AMoRe (16), starting with our previous yeast AHAS structure (Ref. 5; Protein Data Bank accession number 1JSC) as the search model. Rigid body refinement with the CNS software package (17) reduced the Rfactor from 0.476 to 0.424 for data from 6.0 to 2.8 Å resolution. The structural models were checked against the initial 2.8 Å resolution 2Fo-Fc and Fo-Fc electron density maps using the program O (18). Even at the earliest stage of refinement, there was well-defined electron density for both CE and the capping region that will be described later. During all stages of refinement, tight noncrystallographic symmetry restraints were applied to the core regions. Individual B-factors were assigned for all atoms, and an overall anisotropic B-factor correction was applied using the standard protocol in the CNS software package. As well as the two polypeptide structures, 2 FAD molecules, 2 ThDP molecules (1 partially degraded, see below), 2 Mg2+, 2 K+, 2 dithiothreitol molecules, 2 CE molecules, and 832 ordered water molecules were observed in each asymmetric unit. The Rfactor and Rfree for the final structure are 0.163 and 0.205, respectively, and the model has excellent geometry (Table I). The coordinates and structure factors of the yeast AHAS·CE complex have been deposited with the Research Collaboratory for Structural Bioinformatics Protein Data Bank (accession number 1N0H). Figures were generated with LIGPLOT (19), SETOR (20), MOLSCRIPT (21), RASTER 3D (22), WebLab ViewerPro (MSI, San Diego, CA), and INSIGHT2001 (Accelrys).
Mutagenesis, Assay, and Herbicide Inhibition--
Mutations were
introduced by PCR using the megaprimer method (23). AHAS activity and
inhibition constants for imidazolinones and sulfonylureas were
determined using methods described previously (24, 25).
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RESULTS AND DISCUSSION |
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Overall Structure of the AHAS·CE Complex--
Yeast AHAS was
co-crystallized with a sulfonylurea herbicide (CE), and the structure
was solved by molecular replacement using the free enzyme (5) as the
starting model. The AHAS·CE complex has an overall fold that is
similar to that of the free enzyme with the subunits tightly associated
by virtue of numerous noncovalent interactions across the dimer
interface. Each of the two monomers (referred to as A and B) is folded
into three domains of approximately equal size (Fig.
2a), designated as (residues 85-269),
(residues 281-458), and
(residues
473-643). The surfaces of the
- and
-domains in each monomer
form the subunit interface, whereas the
-domains are distal to each
other and play a minor role only in stabilizing the dimer interface.
Each of the domains is constructed around a central, six-stranded,
parallel
-sheet surrounded by
-helices. The
-domain and
-domain have identical topologies, whereas the
-domain consists
of a double Rossmann fold. There are two segments in each monomer where
there is no observable electron density. These correspond to the
N-terminal hexa-histidine tag derived from the expression vector plus
the first 25 residues of the mature protein and to a surface
polypeptide segment connecting the
- and
-domains (Asn-271
to Leu-276 in monomer A and Asn-271 to Thr-277 in monomer B).
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Each dimer has two active sites centered upon the ThDP cofactor. The ThDP is bound at the dimer interface anchored to the protein by Mg2+ and adopts a V-shaped conformation, as is observed in our previous AHAS structure. This conformation is shaped by the side-chain of Met-525, which projects between the thiazolium and pyrimidine rings, forcing them to be oriented at an angle to one another. Two further stabilizing interactions are hydrogen bonds to the pyrimidine ring, from the backbone oxygen of Gly-523 to the 4'-amino group and from the side-chain of the catalytic glutamate (Glu-139) to N1'.
In monomer A, ThDP is missing the S1 and C2 atoms of the thiazolium ring. Loss of C2 from ThDP has been reported previously in the crystal structure of Zymomonas mobilis pyruvate decarboxylase (8). We suggest that formation of the product that we observe (4-{[(4'-amino-2'-methylpyrimidin-5'-yl)methyl]amino}pent-3-enyl diphosphate) results from reactions occurring as a result of the exposure of the crystal to high intensity radiation during synchrotron data collection. Groups containing sulfur are susceptible to this type of damage (26).
Each AHAS monomer contains 1 molecule of FAD. As mentioned earlier, the
role of this cofactor is unclear because the AHAS reaction does not
involve redox chemistry. FAD is in an extended conformation and
interacts mostly with the -domain with the flavin ring pointing
toward the active site (Fig. 2). The overall structure and interactions
of FAD remain largely unaltered in the AHAS·CE complex, compared with
those in the free enzyme. The main change brought about by CE is that
the flavin ring rotates away from CE (Fig. 2b) to avoid a
steric clash between the C7 methyl of FAD and the methoxy carbon atom
of the inhibitor. Superimposing monomer B of the AHAS·CE complex on
to monomer B of the free AHAS structure (Fig. 2b) shows that
the C7 methyl group of the flavin ring (which is at the extremity)
moves by 2.5 Å. When monomer B of the AHAS·CE complex is
superimposed on to monomer A of the free AHAS, the movement of the C7
methyl of the flavin ring is even more pronounced, with the distance
now 4.6 Å.
As mentioned above, Mg2+ anchors ThDP to the enzyme; the
metal ion is coordinated to two phosphate oxygen atoms of the cofactor, the side-chain oxygen atoms of Asp-550 and Asn-577, the backbone oxygen
of Gln-579, and 1 water molecule. This arrangement is similar to that
found in other ThDP-dependent enzymes (6-9) but slightly different from that in the free enzyme (5), where the Gln-579 ligand is
replaced by a water molecule. The displacement of the water in the
present structure is brought about by a reorganization of residues
580-595, as described below. Each monomer contains a single
K+ at the C-terminal end of an -helix that leads away
from the active site, as described previously (5). The yeast AHAS
monomer contains only two cysteine residues, and one of these (Cys-357) is disulfide-linked to dithiothreitol that was present in the crystallization buffer. This nonconserved cysteine is far from the
active site and is unlikely to have any functional importance.
Comparison with the AHAS Structure without CE--
AHAS is a dimer
in crystals of both the free enzyme (5) and in complex with CE. There
are no major differences in the overall fold of both structures, with a
root mean square deviation of 1.15 Å (1059 C atoms) when the
dimeric structures are compared. However, there are two important
changes that are observed in the complex. First, the three domains in
the two monomers of AHAS are brought closer together in the complex,
resulting in a reduction in the volume occupied by the active and
herbicide-binding sites. Second, a capping region (Fig. 2a),
which we define to consist of the 38 C-terminal amino acid residues
650-687 (the "C-terminal arm") and the polypeptide
segment consisting of amino acid residues 580-595 (the "mobile
loop"), becomes ordered, further restricting solvent accessibility to
the active site. This capping region is involved in the formation of a
substrate access channel that is missing in the previous uncomplexed
enzyme structure. The substrate access channel is located at the dimer
interface, and its inner face is formed by residues from all the three
domains (Fig. 3a). The
reaction center C2 atom of ThDP is positioned at the bottom of this
channel, about 15 Å from the protein surface. In the structure without
CE, the entire thiazolium ring of ThDP is solvent-accessible. As a
result of the presence of the additional capping region in the current
structure, most of ThDP is buried, and only the C2 atom of ThDP would
be readily accessible to solvent.
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As can be observed in Fig. 2, the C-terminal arm in the AHAS·CE
complex adopts well-defined elements of secondary structure with
residues 653-655 and 664-666 forming a small anti-parallel -sheet,
whereas the segment 669-683 forms an
-helix. As shown in Fig.
2a, residues 653-666 of this arm reach across from the
-domain and attach to the
-domain. Phe-664 fits into a pocket on
the surface of the
-domain that is bordered by Ala-358, Leu-362, and
the aromatic ring of Tyr-458. A small polar surface on the tip of the
arm forms stabilizing interactions between the arm and the
-domain,
with an ion pair between Glu-663 and Lys-387, a hydrogen bond between
Asp-662 and Gln-365, and a hydrogen bond between the carbonyl oxygen of
Gly-658 and the side-chain of Asn-384. In addition, a series of van der
Waals contacts involving Val-649, Val-651, Pro-653, Met-654, Val-655,
Leu-661, and Phe-667 are made between the C-terminal arm and the
-domain. This feature, along with the enzyme-herbicide interactions,
may provide an explanation for the overall tightening of the structure
of the AHAS·CE complex. A further difference involves the mobile loop
(residues 580-595); in the AHAS·CE complex, this region is organized
into an
-helix (residues 580-589) and coil (residues 590-595)
structure (Fig. 2b). In our previously published structure
of yeast AHAS in the absence of CE (5), the entire mobile loop is
completely disordered in one monomer but traceable as a random coil in
the other (Fig. 2b). The corresponding region in related
ThDP-dependent enzymes (6-9) is folded in an
-helix/coil structure closing over the active site, similar to that
observed in the AHAS·CE complex. Apart from these major changes, some
other differences are observed in the AHAS·CE complex, most notably
the movement of the flavin ring of the FAD, mentioned previously, and
also the conformation and position of the side-chains of Arg-380 and
Met-354 have been altered significantly to optimize interactions with
CE.
Two pieces of evidence suggest that the structural changes observed in the AHAS·CE complex are herbicide-induced and are not imposed by crystallization. First, attempts to co-crystallize AHAS and CE under the same conditions as the enzyme without CE were not successful. Similarly, conditions for crystallizing the complex do not yield crystals when the inhibitor is omitted. Second, diffraction data on existing crystals of AHAS soaked with CE (and other herbicidal inhibitors) do not show interpretable electron density for the herbicide.
The C-terminal arm does not have an equivalent in the closely related
ThDP-dependent enzymes pyruvate oxidase, pyruvate
decarboxylase, or benzoylformate decarboxylase. All have an -helix
near the C terminus, but none of them has the extended loop reaching
across to interact with the
-domain. The extended loop structure may be related to another distinctive feature of AHAS: it is the only one
of these four enzymes that possesses a regulatory subunit. The binding
site for the regulatory subunit on AHAS has not been determined, but we
speculate that the extended loop may form this binding site, possibly
by acting as a clamp that wraps around the regulatory subunit.
Location and Conformation of CE--
CE is bound in the substrate
access channel of AHAS (Fig. 3b). An electron density map
defining the conformation of CE is shown in Fig.
4. In contrast to the conventional
extended conformation in which sulfonylureas are usually represented
(Fig. 1a), CE is folded at the sulfonyl group with the two
rings almost orthogonal to one another in two planes, with the ethyl
side-chain extended parallel to the heterocyclic ring. The sulfonyl
group and the attached aromatic ring are situated at the entrance to
the substrate access channel, with the rest of the CE inserting into
the channel (Fig. 3, a and b). Thus, CE
completely blocks access to the active site, inhibiting AHAS by this
mechanism.
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CE is an extremely potent inhibitor of AHAS, with an inhibition
constant in the low nanomolar range. The sulfonylurea-binding site is
located at the dimer interface and is in the vicinity of the active
site and the flavin ring of FAD. CE forms mainly hydrophobic contacts
with the protein and FAD (Fig. 5),
interacting with residues from both monomers and all three
domains and with the C7 methyl group of FAD. In addition, the
sulfonylurea bridge of the herbicide forms four hydrogen bonds with two
amino acid residues Lys-251' and Arg-380. Most of these contacts are
with residues where mutation results in herbicide-resistant variants (see below). Two amino acids (Met-582 and Trp-586) within the mobile loop that are known herbicide resistance sites exist in two
conformations. Both residues are in direct contact with CE, and the
alternate conformations of the side-chains are held in place by the
bound herbicide.
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Inhibition of AHAS by sulfonylureas is a time-dependent
process (2, 10, 13). The initial inhibition that is observed immediately upon mixing the enzyme with substrate in the presence of
these inhibitors becomes progressively stronger, taking tens of minutes
to develop fully. This time dependence does not appear to be due to
slow binding per se (2) because preincubation of the enzyme
with sulfonylureas does not promote inhibition. It appears that ongoing
catalysis is required for inhibition to develop, and this is consistent
with the fact that inhibition is not competitive with the substrate
(10, 27). This, in turn, leads to the suggestion (28) that
sulfonylureas combine better with the enamine/-carbanion reaction
intermediate that is formed after decarboxylation of the first molecule
of pyruvate. If this hypothesis is correct, then it follows that, in
the AHAS·CE complex, there should be a cavity in the active site that
would be able to accommodate the intermediate. Examination of the
structure reveals the existence of such a cavity (Fig.
6) occupied by a single water molecule
only. The intermediate can be modeled into the structure with no
unfavorable interactions and a stabilizing hydrogen bond to the
4'-amino of ThDP. There is another consequence of the hypothesis that
sulfonylureas combine better with the enzyme containing the
enamine/
-carbanion reaction intermediate than with the enzyme at
rest, the structure determined in the present report. To account for
the stronger inhibition by sulfonylureas during AHAS catalysis, there
should be additional interactions between the herbicide and the enzyme
when it contains the reaction intermediate. To test this hypothesis
experimentally would require crystallization of AHAS trapped as the
reaction intermediate (or a close analog) together with the herbicide.
The corresponding intermediate in transketolase has been trapped by
adding a donor substrate that produces this complex while omitting the
acceptor substrate that reacts with the intermediate (31). With AHAS, this strategy is not possible because pyruvate, the substrate required
to generate the enamine/
-carbanion, is also the acceptor substrate
with which it reacts.
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Contacts with Herbicide Resistance Sites-- Herbicide-resistant variants of AHAS have been identified in the enzyme from various microorganisms and plants. In yeast AHAS, mutations at 10 separate sites have each been shown to confer sulfonylurea insensitivity (29), and nine of these residues make direct, mainly hydrophobic, contacts with CE (Fig. 5). Only Phe-590 is not in direct contact with CE but this overlies Trp-586, which is locked into its two alternate conformations by being sandwiched between Phe-590 and the pyrimidine ring of CE. Other contacts to CE are with Val-191', Phe-201', Arg-380, and Met-582. Of these, the equivalent residues to both Val-191 and Met-582 have been shown to confer sulfonylurea resistance when mutated in Escherichia coli AHAS isoenzyme II (27, 30). The effect of mutation at Phe-201 and Arg-380 has not been tested to our knowledge.
Based on the structure of the AHAS·CE complex, it is easy to imagine how mutation of the contact residues might result in insensitivity to CE. We have constructed and performed detailed characterization of a series of herbicide-resistant AHAS mutants, and the results will be published elsewhere. Here we present some examples of AHAS mutations leading to sulfonylurea insensitivity. Mutations of Trp-586 are commonly identified in laboratory and field isolates, leading to strong resistance to sulfonylureas as well as to other classes of AHAS inhibitor. The mutation W586L in yeast AHAS results in a massive 6250-fold reduction in CE sensitivity. Our AHAS·CE structure shows that this amino acid is involved in ring-stacking interactions with the pyrimidine ring of CE (Fig. 3c). Mutation to a smaller and nonaromatic amino acid will greatly disrupt the interaction, consistent with the observed very large increase in the inhibition constant. Gly-116 lies between the pyrimidine ring and the ethyl side-chain (Fig. 3d), oriented so that any amino acid substitution would create steric clashes with CE unless there are compensating structural alterations of the protein. We have measured the apparent Ki of the yeast AHAS G116S mutant and shown that this variant is 1000-fold less sensitive to CE than the wild-type. A third example is Lys-251 (Fig. 3e); here the contact is a hydrogen bond between the side-chain amino group and one of the sulfonyl oxygen atoms. Substitution of Lys-251 by Thr, which would prevent this hydrogen bond from forming, results in 23-fold resistance to CE. The agreement between the enzyme structure (determined in the absence of substrate) and the effects of mutations on CE inhibition (measured in the presence of substrate) suggests that the location and orientation of CE are not changed drastically during catalysis.
Binding of Other Herbicides--
Yeast AHAS is inhibited by a
range of sulfonylurea herbicides, with apparent Ki
values ranging from 3.3 nM (CE) to 127 nM
(chlorsulfuron). It is likely that each binds in a similar manner to
CE, although this has yet to be verified experimentally. The binding of
imidazolinone herbicides is more problematic, and these compounds are
substantially weaker inhibitors than the sulfonylureas, with apparent
Ki values in the 1-10 mM range. The
considerable structural differences between the sulfonylureas (Fig.
1a) and the imidazolinones (Fig. 1b) make it
unlikely that they would be able to form the same interactions,
although we presume that both types of inhibitor bind in the substrate
access channel. It is known that many AHAS mutations result in
cross-resistance to both families of herbicide, but some are rather
specific, resulting in resistance to one family of herbicides but not
to the other. For example, P192S results in 65-fold resistance to CE
but has no effect on imidazolinone sensitivity. Conversely, the
mutation M354V has greater effects on imidazolinone sensitivity than CE sensitivity. Determination of the structure of yeast AHAS in complex with other sulfonylureas or imidazolinones will thus be of considerable interest.
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ACKNOWLEDGEMENTS |
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Some yeast AHAS mutants were constructed and characterized in this laboratory by Hongqi Yu. Sulfonylurea and imidazolinone herbicides were gifts from Dr. S. Gutteridge (DuPont) and Dr. B. K. Singh (BASF), respectively. We thank Harry Tong, Gary Navrotski, and Keith Brister for assistance at Beam Line 14D, Advanced Photon Source, Argonne National Laboratory. Data collection was performed with support from the Australian Synchrotron Research Program, which is funded by the Commonwealth of Australia under the Major National Research Facilities Program.
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FOOTNOTES |
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* This work was supported by grants from the Australian Research Council (to R. G. D. and L. W. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1N0H) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
To whom correspondence should be addressed: Dept. of Biochemistry
and Molecular Biology, The University of Queensland, Coopers Rd., St.
Lucia, Brisbane QLD 4072, Australia. Tel.: 61-7-3365-4615; Fax:
61-7-3365-4699; E-mail: Ronald.Duggleby@mailbox.uq.edu.au.
Published, JBC Papers in Press, December 20, 2002, DOI 10.1074/jbc.M211648200
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ABBREVIATIONS |
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The abbreviations used are: AHAS, acetohydroxyacid synthase; CE, chlorimuron ethyl; FAD, flavin adenine dinucleotide; ThDP, thiamine diphosphate.
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