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J. Biol. Chem., Vol. 282, Issue 15, 11446-11455, April 13, 2007
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12
13
From the
Pioneer Hi-Bred International, Redwood City, California 94063 and the Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637
Received for publication, November 2, 2006 , and in revised form, February 1, 2007.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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To develop gat as a transgene for glyphosate resistance in crops, we subjected the three B. licheniformis enzymes to 11 rounds of gene shuffling and obtained optimized variants with up to a 4,500-fold increase in catalytic efficiency (kcat/Km) relative to the native enzyme. This was achieved through a combination of improvements in kcat (190-fold) and Km,GPJ (24-fold). When introduced into plants, optimized gat genes confer robust tolerance to glyphosate (1).
To gain insight into the molecular mechanism of glyphosate N-acetylation by GAT, we determined the structure of a 7th round GAT variant (termed R7 GAT) in ternary complex with AcCoA and the competitive inhibitor 3-phosphoglycerate (3PG) and in binary complex with oxidized CoA. We also carried out a detailed kinetic analysis of the activity of native, optimized, and site-directed mutants of GAT against a panel of substrate analogs. Together, these data allow us to propose a detailed mechanism for glyphosate N-acetylation by GAT. Furthermore, comparison of the native and optimized enzymes provides insight into the structural basis of the activity enhancements achieved through gene shuffling.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Mutagenesis, Protein Expression, and Purification—Native and variant GAT enzymes were prepared as described (1). Briefly, gat genes were expressed in Escherichia coli from a pQE80 vector (Qiagen), modified to remove the His6 tag. GAT was purified from cell lysates by affinity chromatography on coenzyme A-agarose (Sigma) and gel filtration chromatography on Superose 75 (Amersham Biosciences).
Site-directed mutagenesis of the R7 gat gene in pQE80 was performed using a QuikChange site-directed mutagenesis kit (Stratagene). Oligonucleotide pairs containing the substituted nucleotides were purchased from Operon Biotechnologies, Inc. All resulting plasmids were sequenced to verify that only the intended mutation was present in the coding region. Double mutants were obtained by successive application of the site-directed mutagenesis protocol.
Protein concentrations were calculated from measurements of absorbance at 205 nm (Emg/ml = 30.5) after buffer exchange (NAP-5 column, Amersham Biosciences) into 50 mM Na2SO4. The extinction coefficient was determined by the procedure of Scopes (5), in which extinction, cm-1 (mg/ml)-1 = 27 + 120 (A280/A205).
Kinetic Analysis—Apparent Km values for glyphosate and the compounds in Table 3 were determined as described previously (4) in a continuous spectrophotometric assay in which cleavage of the thioester bond of AcCoA is monitored at 235 nm in a Spectramax Plus384 plate reader (Molecular Devices, Sunnyvale, CA). Reaction mixtures contained 25 mM Hepes, pH 6.8, 10% ethylene glycol, varied concentrations of acceptor substrate and saturating AcCoA (167 µM). The Spectramax software was programmed to convert change in absorbance to reaction velocity, µM/min, and to return parameters of the Lineweaver-Burk transformation of the Michaelis-Menten equation. The kcat value was calculated from Vmax and the concentration of GAT. The apparent Km value for AcCoA was determined from initial rate measurements at saturating glyphosate, using mass spectrometric detection of NAG, as described (4), and the Lineweaver-Burk method.
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For R7 and R11 GAT, Ki values for the competitive inhibitors NAG, CoA, 3PG, and phosphoenolpyruvate were determined from slope re-plots of double-reciprocal plots in which AcCoA was fixed at 167 µM; the glyphosate was varied, and inhibitor was supplied at a selection of fixed concentrations. For native GAT, the Ki value for CoA was also determined as above. For all other enzyme and inhibitor combinations, Ki values were determined from a single concentration of inhibitor using the equation Km,app = Km + Km/Ki [I]. Inhibitor concentrations were as follows: 2 mM NAG, 50 µM 3PG, 10 µM phosphoenolpyruvate, 15 µM phosphonopyruvate with native GAT; 20 µM phosphonopyruvate with R7 and R11 GAT; and 5 µM phosphonoacetate and 2 mM phosphonobutyrate with all enzymes.
Crystallization and Data Collection—Purified R7 GAT was buffer-exchanged and concentrated to 10 mg/ml in 5 mM Hepes, pH 7.5. For crystallization the protein was incubated in the presence of 2 mM AcCoA alone or with 2 mM AcCoA and 20 mM 3PG. Crystals were obtained overnight by hanging drop vapor diffusion against a crystallization solution containing 100 mM NaOAc, pH 4.6, 150-300 mM ammonium sulfate, and 20-25% PEG4000. The crystals were mounted in nylon loops, after first transferring them to cryoprotectant comprising 100 mM NaOAc, pH 4.6, 25% PEG4000, and 20% glycerol supplemented with 2 mM AcCoA and 250 mM ammonium sulfate (binary complex) or 2 mM AcCoA, 50 mM 3PG, and 50 mM ammonium sulfate (ternary complex). Data were measured at 100 K at beamline 5.0.2 of the Advanced Light Source (ALS, Berkeley, CA) using an ADSC Q210 CCD detector. Although the crystals diffract to high resolution, as evidenced by the low Rsym and high 
I/
I 
values in the highest resolution shell, the detector geometry limited the data collection to 1.6 Å. All data were processed using MOSFLM (6) and programs from the CCP4 suite (7). Data collection and refinement statistics are summarized in Table 1.
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30% occupancy) active site configuration in which 3PG is replaced by a sulfate ion and an additional water molecule. We did not observe convincing electron density for the acetyl group of AcCoA in the binary complex structure. Based on careful inspection of Fo - Fc difference maps, we modeled the cofactor as oxidized CoA, although we cannot exclude the possibility that the bound species is AcCoA with a rotationally disordered acetyl group. No evidence for transfer of the acetyl group to the thiol of Cys-108 was observed, either kinetically or in the electron density maps of either structure. Refinement was carried out in REFMAC5 (9), and COOT (10) was used for model building. The use of anisotropic B-factors in the final stage of refinement was supported by a >1% drop in Rfree and Rcryst for both data sets. All amino acids except for the N-terminal methionine, which is disordered, are included in the final models. PyMOL was used to generate all structure figures (11). Atomic coordinates and structure factors have been deposited in the PDB with accession numbers 2JDC (binary complex) and 2JDD (ternary complex). |
RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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3PG-binding Site—The competitive inhibitor, 3PG, binds in a solvent-exposed cleft adjacent to the cofactor and the V-shaped wedge formed by 
4 and 5. The binding site is flanked on one side by the region extending from the C-terminal portion of 
1 into 2b and on the other side by the 6-7 loop (Fig. 2A). These flanking regions are among the least conserved structural features within the GNAT superfamily, a reflection of the fact that these enzymes bind to a diverse set of substrates. The binding site is positively charged and surrounded by an otherwise electronegative surface (Fig. 2B). A total of 8 amino acids, 1 water molecule, and the acetyl group of AcCoA interact directly (<4 Å) with the inhibitor (Fig. 2C). The hydrophobic side chains of Leu-20, Phe-31, and Val-135 all contact 3PG, as does the main chain nitrogen of Gly-74, which forms a hydrogen bond with the carboxylate of 3PG. The majority of contacts, however, are made between charged groups, including side chain interactions with the phosphate end (Arg-21, Arg-111, and His-138) and with the carboxylate end (Arg-21 and Arg-73) of 3PG. Of particular note is a short, 2.46-Å hydrogen bond between the N-
of His-138 and a phosphate oxygen of 3PG.
In contrast with the conformation observed in the binary complex, which contains a sulfate ion bound in the substratebinding site, residues in the 6-7 loop are displaced by up to 1.5 Å away from the active site in the ternary complex. This loop possesses the highest average B-factors in the ternary complex structure. In addition, side chains of residues lining the opposite face of the binding site, including Arg-21 and Arg-73, are displaced by up to 1.3 Å away from the active site to accommodate 3PG binding.
Certain features of the GAT structure appear to link the substrate- and cofactor-binding sites. Three well ordered water molecules are buried at the interface where 1, 2a, and 2b pack against the central -sheet (Fig. 2A). The most deeply buried of these waters makes a series of hydrogen bonds to residues from three distinct regions of the enzyme: Arg-16, located at the C-terminal end of 1; Ser-55, located in the center of 3; and the backbone carbonyl of Gly-74, located in the -bulge region of 4. Ser-55 is conserved in the GAT-like family of N-acetyltransferases, as is Arg-16, which forms a buried salt bridge with another conserved residue Asp-34 (on 2b). The proximity of these residues to the cofactor-(Gly-74) and substrate-binding sites (Leu-20, Arg-21, Phe-31, Arg-73, and Gly-74) suggests that the network of hydrogen bonds formed between 3, 4, 2a, and 2b is required for optimal binding of both ligands. Indeed, substitution of Asp34 with alanine in an 8th round GAT variant decreased Km values for both AcCoA and glyphosate by 44- and 13-fold, respectively (data not shown).
Cofactor and Substrate Specificity for Native GAT and Optimized Variants
The steady-state kinetic parameters of native and optimized GAT enzymes used in this study are summarized in Table 2.
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Acceptor Substrate Specificity—To define the range of sizes and functional groups that are preferred in substrates utilized by native, R7, and R11 GAT, we tested a series of compounds related to glyphosate (Table 3). Aminomethylphosphonate (AMPA), a glyphosate analog lacking a carboxyl group, was acetylated with nearly the same kcat value as glyphosate by the native, R7, and R11 enzymes, but its Km values are much higher than those of glyphosate. Relative to glyphosate, the Km value for AMPA is 3.6-fold greater with native GAT, 17-fold greater with R7, and 133-fold greater with R11. Interestingly, N-methyl-AMPA had Km values similar to those of AMPA, but kcat values were 27- to 50-fold lower, suggesting steric hindrance from the methyl group. Glycine and N-methylglycine (sarcosine) were tested to probe the effect of removing the phosphonate group from glyphosate. Even when supplied at 100 mM, essentially no activity was observed with any of the three GAT variants. The contrasting effects obtained from removal of the phosphonate group compared with removal of the carboxyl group show that the former provides more binding energy for catalysis with glyphosate.
The only compound we tested that was acetylated appreciably by native GAT was D-2-amino-3-phosphonopropionate (D-AP3), exhibiting a kcat/Km of 1500 min-1 mM-1. Activity with D-AP3 was less with R7 and R11 (Table 3). Activity with the L-isomer was very poor with the native enzyme and almost undetectable with the optimized variants. The DL form of the next longer compound, DL-2-amino-4-phosphonobutyrate (DL-AP4), showed only slight activity at 100 mM with all enzymes, and the longest, DL-2-amino-5-phosphonopentanoate (DL-AP5), was devoid of reactivity (see below).
We also tested a series of compounds for their ability to inhibit glyphosate N-acetylation (Table 3). NAG inhibited each enzyme competitively with glyphosate. Ki values decreased in parallel with Km,GPJ and were 1.3 mM (native), 0.35 mM (R7), and 0.06 mM (R11). Despite being a poor substrate, L-AP3, was a potent inhibitor of both R7 and R11, with Ki values of 5 and 10 µM, respectively (Table 3). The next longer compound, DL-AP4, was a poor inhibitor of native GAT but bound 53-fold more tightly to R11 than to native GAT. The longest compound, DL-AP5, did not inhibit native GAT and only weakly inhibited R7 and R11. A similar trend was observed with the competitive inhibitors 3PG and phosphoenolpyruvate (PEP). The shorter of the two compounds, PEP, showed lower Ki values than 3PG for native, R7, and R11, but the fold difference decreased throughout the optimization as follows: 12-fold tighter with the native enzyme, 5.3-fold with R7, and only 2.4-fold with R11. Similar results were obtained with the PEP analog, phosphonopyruvate. The shortest compound tested, 2-phosphonoacetate was a potent inhibitor of the native, R7, and R11 enzymes.
Kinetic Analysis of Site-directed Mutants
Glyphosate Binding—In the crystal structure of R7 GAT bound to AcCoA and 3PG, there are 8 residues located within 4 Å of the inhibitor. To examine the role of these residues in binding to glyphosate, we constructed a series of point mutants in R7 GAT and determined their kinetic parameters (Table 4). Mutation of Arg-111, Arg-73, or Arg-21 to alanine resulted in a 170-250-fold increase in Km,GPJ. Likewise, a significant increase in glyphosate Km was observed for H138A (44-fold) and the more conservative L20I (32-fold) mutant.
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To identify ionizable groups that are relevant to the catalytic mechanism, we measured the pH dependence of kcat for R7 GAT. As shown in Fig. 3A, the pH profile indicates that kcat depends on the ionization of two groups. The first must be deprotonated for activity, and likely corresponds to a catalytic base with a pK value of 5.7. The second must be protonated for activity, and likely corresponds to a catalytic acid with a pK value of 7.5.
Given its sequence conservation (Fig. 1) and its location 5.5 Å from the presumptive position of the nitrogen atom of glyphosate (pKa of 10.3 (3)) (Fig. 2C), His-138 is the most likely amino acid to function as a general base. Replacing His-138 with alanine in R7 GAT caused a 110-fold decrease in kcat relative to the R7 GAT enzyme (Table 4). Substituting alanine for any of the conserved active site arginines, Arg-111, Arg-21 and Arg-73, reduced kcat more modestly, by 26-, 4.3-, and 1.3-fold, respectively.
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Shuffling Changes—There are a total of 21 amino acid substitutions in R7 GAT resulting in a 200-fold increase in kcat and a 5.4-fold decrease in Km,GPJ relative to the native enzyme (Table 2). This sequence variation is distributed across the entire structure (Fig. 2D), with only 4 of the 21 substitutions located within 5 Å of either AcCoA or 3PG (Figs. 1 and 2C). To examine the effect of individual shuffling changes on catalysis, we generated four site-directed mutants in which these active site residues (Phe-31, Ala-114, Thr-132, and Val-135) in the R7 enzyme were changed back to the native sequence. Surprisingly, the individual revertants had little impact on kcat, with 3 of the 4 revertants actually showing slight improvement (Table 4). In contrast, A114V (13-fold) and V135I (6.3-fold) showed a significant increase in Km,GPJ relative to R7 GAT. The other two revertants, T132I and F31Y, show more modest increases in Km,GPJ (Table 4). Most significant is that in all cases, the variant residue possesses a smaller side chain than the native residue.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Based on the stereochemical similarity of the compounds, we infer that glyphosate binds in the same location and orientation as 3PG, with its carboxyl group in contact with Arg-73 and Arg-21, and its phosphonyl group in contact with His-138, Arg-21, and Arg-111. Site-directed alanine mutants implicate each of these residues in glyphosate binding. In particular, alanine substitutions at any of the active site arginines dramatically increase Km,GPJ by 170-250-fold (Table 4).
With a pKa of 10.3 (3), the glyphosate amine is largely protonated at physiological pH and must be deprotonated prior to acetyl transfer. In GAT, the same basic residues implicated in glyphosate binding also contribute to a positively charged active site (Fig. 2B). As proposed in the case of the yeast histone acetyltransferase HpaII (17), this net positive electrostatic potential is likely to lower the pKa value of the substrate amine upon substrate binding. However, individual alanine substitutions constructed at each of the basic GAT residues reveal significant and differential effects on kcat. Although alanine mutations at any of the active site arginines reduce kcat between 1.3- and 26-fold, mutation of His-138 to alanine results in a 110-fold reduction in kcat (Table 4). A decline of this magnitude is consistent with that expected for loss of a catalytic base (18-20), and it suggests that His-138 plays a specific catalytic role during the reaction.
All GAT variants that we tested, including the three native B. licheniformis GAT enzymes (1), contain histidine at position 138 and exhibit an optimal kcat at pH 6.8, as shown for R7 in Fig. 3A. The pH-rate analysis of R7 GAT with glyphosate (Fig. 3A) suggests the presence of one or more ionizable groups in the enzyme-substrate complex with a pK1 of 5.7 that must be deprotonated for maximal activity, and we attribute this to His-138 (solution pKa 6.0) and to the phosphonate group of glyphosate (solution pKa 5.7).
In the ternary complex His-138 is located 5.5 Å from C-3 of 3PG, the atom corresponding to the nitrogen of glyphosate (Fig. 2C). This is too far for His-138 to function directly as a catalytic base. In the true substrate complex subtle differences in the conformation of glyphosate (relative to 3PG) or in the position of His-138 might bring the substrate amine closer to His-138. Alternatively, the specific effect of His-138 on kcat could be mediated by water molecules in the active site, as observed in other members of the GNAT family (15, 16). However, none of the active site waters observed in the crystal structure are located within hydrogen bonding distance of the His-138 side chain.
We favor an alternative mechanism by which His-138 catalyzes substrate deprotonation, shown in Fig. 3B. In the crystal structure, determined at pH 4.6, His-138 forms a short 2.46-Å hydrogen bond with one of the phosphate oxygens of 3PG. Similar short hydrogen bonds are observed in the active sites of a number of enzymes, and they are proposed to play a variety of significant roles in catalysis (21-23). At physiological pH, the His-138 side chain and the glyphosate phosphonate group are likely in a deprotonated state. Upon substrate binding we propose that a proton from the secondary amino group of glyphosate is stabilized on a phosphonate oxygen atom, resulting in formation of a strong hydrogen bond between His-138 and glyphosate (Fig. 3B) and activation of the substrate amine. This substrate-assisted proton transfer mechanism is consistent with the observed pH dependence of kcat (Fig. 3A) and explains the dual role of His-138 in substrate binding and as a catalytic base.
Polarization of the AcCoA carbonyl thioester and stabilization of the tetrahedral intermediate formed after attack by the substrate amine lone pair is typically performed in GNAT enzymes by a pair of main chain amide hydrogens (15, 16). In R7 GAT, the main chain amides of residues Gly-74 and Met-75, part of the structurally conserved -bulge in 4, are positioned to fulfill this role (Fig. 2C).
In some members of the GNAT superfamily an active site residue functions as a general acid to protonate the thiolate anion that forms on CoA upon collapse of the tetrahedral intermediate (15, 16). In GAT, the hydroxyl group of Tyr-118 is located within 3.3 Å of the sulfur atom of CoA, where it is perfectly positioned to perform this function. Mutating Tyr-118 to phenylalanine resulted in an 17-fold drop in kcat at pH 6.8 (Table 4) and eliminated the basic limb of the pH versus kcat plot (Fig. 3A), suggesting that Tyr-118 should be in the neutral form for optimal GAT activity. Indeed, kcat for R7 at pH 8.5, where Tyr-118 would largely be de-protonated, is nearly the same as for R7-Y118F. The rate acceleration we attribute to Tyr-118 (17-fold) is typical for acid-base catalysts (18) and is similar to that seen in other members of the GNAT superfamily (14, 19).
Substrate Specificity and Optimization for Glyphosate
Substrate Specificity—The physiological role and natural substrates of the bacterial GAT-like enzymes (Fig. 1) are unknown. The most active compound we tested with native GAT is the D-isomer of AP3, a 4-atom (main chain) molecule that is acetylated with a kcat/Km value of 1500 min-1 mM-1. Although it is significantly more active than any of the aminocontaining compounds we tested against native GAT, D-AP3 is a relatively poor substrate compared with the substrates of other N-acetyltransferases (4, 24-26). Furthermore, although AP3 is a naturally occurring phosphonate, found in Tetrahymena and the soft coral Zoanthus (27-29), there is no defined physiological role for the D-isomer of AP3.
Analysis of the structure-activity relationship for the series of inhibitors tested against the native and optimized GAT enzymes reveals two general trends as follows: 1) native GAT binds shorter ligands (chiefly 3 and 4 atoms in the main chain) more tightly than longer ones, and 2) progressive optimization for glyphosate activity is accompanied by improved binding to longer ligands (up to 5 atoms in the main chain) and retained binding to shorter ligands (Table 3). For example, the 3- and 4-atom main chain compounds (e.g. 2-phosphonoacetate, phosphonopyruvate, and PEP) are all significantly better inhibitors (Ki 10 µM) of native GAT than are the 5- and 6-atom main chain compounds (Ki values ranging from 140 µM to no measurable inhibition). After optimization for the 5-atom main chain substrate glyphosate, the 5- and 6-atom main chain compounds (e.g. NAG, DL-AP4, and DL-AP5) bind more tightly (up to 50-fold) to R11 than to native GAT, whereas the shorter compounds remain potent inhibitors of the native and optimized enzymes.
The active site of GAT dictates not only the acceptable size of a substrate but also its stereochemical configuration. This is best illustrated by AP3. Although the D-isomer is a substrate for each enzyme, the L-isomer is a potent inhibitor of R7 and R11.
Taken together with previous results (4), the structure-activity data indicate a narrow substrate range for native GAT and variants optimized for acetylation of glyphosate. Appreciable activity requires an amine-containing compound with a phosphonyl or phosphoryl and a carboxyl group with a main chain length of 5 or fewer atoms. Of the numerous compounds we tested (Table 3) (4), those that lie outside these parameters all fail to exhibit appreciable activity with native GAT or any variant.
It is interesting to note that the activity for D-AP3 drops by less than 5-fold in R11 relative to the native enzyme, whereas glyphosate activity increases by 4,500-fold. Thus, the original GAT activity against D-AP3 is robust to the large number of changes accumulated in R11 GAT during the course of the optimization for glyphosate. This is consistent with other experiments in which optimization of a weak starting activity is driven by mutations that have little effect on the original (strong) activity but a dramatic effect on the optimized activity (30).
Glyphosate Optimization—The 1100-fold increase in glyphosate N-acetylation by R7 GAT is achieved through a combination of improvements in kcat (200-fold) and in Km,GPJ (5.4-fold). There are 21 amino acids differences (out of 146) between R7 GAT and the native enzyme (Fig. 2D, yellow spheres). Strikingly, none of these changes occur at sites comprising the catalytic machinery. Indeed, only four of the changes are located within 5 Å of the cofactor or 3PG (Fig. 2D, blue spheres). In almost all of these cases, Y31F, V114A, I132T, and I135V, the optimized enzyme contains a smaller side chain than that present in the native enzyme, consistent with the kinetic data suggesting that GAT is optimized to accommodate a larger substrate, glyphosate.
Single-site revertants constructed at each of the four active site variable positions reveal three that significantly increase Km,GPJ (Table 4). In particular, a 6.2-fold increase in Km,GPJ is observed for the R7-V135I revertant. This substitution alone accounts for the entire 5.4-fold improvement in Km,GPJ observed in the R7 enzyme. The inability of glyphosate to inhibit the glyphosate-insensitive G96A mutant of EPSP synthase from E. coli arises from a van der Waals clash between the methyl group of Ala-96 and the phosphonate group of glyphosate (31). In the crystal structure of GAT, Val-135 is in an analogous position, just 3.5 Å from the glyphosate phosphonate group. It appears that gain-of-function substitutions in the optimized R7 GAT serve, in part, to relieve similar steric clashes in the substrate-binding pocket of native GAT.
The present results offer little insight into how the 200-fold increase in kcat was achieved in the optimized R7 enzyme. Single-site revertants constructed at each of the four active site positions reveal minimal effects on kcat. Substitutions located distal to the active site (Fig. 2D, yellow spheres) may remodel the active site through subtle rearrangements that influence the enzyme structure and flexibility (32-35) to speed up the ratelimiting step of the overall reaction.
The outcome of our optimization experiments with GAT is an enzyme with activity against glyphosate that is comparable with that of other N-acetyltransferases for their natural substrates. This includes another herbicide resistance enzyme, phosphinothricin acetyltransferase, which acetylates phosphinothricin (PPT) with a kcat/Km,AcCoA of 6 x 104 M-1 s-1 and a kcat/Km,PPT of 3 x 105 M-1 s-1(4). The kcat/Km of the R11 GAT enzyme for AcCoA and glyphosate is 2 x 107 and 5 x 105 M-1 s-1, respectively. In contrast, the diffusion-limited maximal value for kcat/Km is 109 M-1 s-1 (18). Thus, despite the dramatic improvement in activity with glyphosate, GAT is far from the limits of optimization.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
1 Both authors contributed equally to this work.
2 To whom correspondence may be addressed. Tel.: 650-298-3556; Fax: 650-701-1258; E-mail: dan.siehl{at}pioneer.com.
3 To whom correspondence may be addressed. Tel.: 773-834-2292; Fax: 773-702-0439; E-mail: bkeenan{at}uchicago.edu.
4 The abbreviations used are: EPSP, 5-enolpyruvylshikimate 3-phosphate; GAT, glyphosate N-acetyltransferase; GNAT, GCN5-related N-acetyltransferase; AcCoA, acetyl coenzyme A; CoA, coenzyme A; GPJ, glyphosate; 3PG, 3-phosphoglycerate; NAG, N-acetylglyphosate; 3PG, 3-phosphoglyceric acid; AP3, 2-amino-3-phosphonopropionic acid; AP4, 2 amino-4-phosphonobutyric acid; AP5, 2-amino-5-phosphonopentanoic acid; PEP, phosphoenolpyruvate; AMPA, aminomethylphosphonate; r.m.s.d., root mean square deviation; PDB, Protein Data Bank.
5 OptimumTM GATTM is the trademark name that Pioneer Hi-Bred International, Inc., uses to identify its glyphosate ALS tolerance trait.
6 The GenBankTM accession numbers for the sequences contained herein are as follows: B. licheniformis ST401 GAT, AX543338; R7 GAT, AY597417; R11 GAT, AY597418; B. subtilis YITI, CAA70664; Bacillus cereus GNAT, NP_830505; Bdellovibrio bacteriovorus GNAT, NP_967337; Chloroflexus aurantiacus GNAT, ZP_00018467; Exiguobacterium sibiricum GNAT, ZP_00538687; Listeria inocua GNAT, CAC98097.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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) and R7-Y118F (
) GAT. The pK1 and pK2 values shown on the graphs refer to the acidic and basic limbs of the profiles, respectively. See "Experimental Procedures" for experimental conditions and theoretical fits. B, proposed chemical mechanism for N-acetylation of glyphosate by GAT.