Redesigning the Substrate Specificity of an Enzyme by Cumulative Effects of the Mutations of Non-active Site Residues*

Shinya Oue, Akihiro Okamoto, Takato Yano, and Hiroyuki KagamiyamaDagger

From the Department of Biochemistry, Osaka Medical College, Takatsuki, Osaka 569-8686, Japan

    ABSTRACT
Top
Abstract
Introduction
References

Directed evolution was used to change the substrate specificity of aspartate aminotransferase. A mutant enzyme with 17 amino acid substitutions was generated that shows a 2.1 × 106-fold increase in the catalytic efficiency (kcat/Km) for a non-native substrate, valine. The absorption spectrum of the bound coenzyme, pyridoxal 5'-phosphate, is also changed significantly by the mutations. Interestingly, only one of the 17 residues appears to be able to contact the substrate, and none of them interact with the coenzyme. The three-dimensional structure of the mutant enzyme complexed with a valine analog, isovalerate (determined to 2.4-Å resolution by x-ray crystallography), provides insights into how the mutations affect substrate binding. The active site is remodeled; the subunit interface is altered, and the enzyme domain that encloses the substrate is shifted by the mutations. The present results demonstrate clearly the importance of the cumulative effects of residues remote from the active site and represent a new line of approach to the redesign of enzyme activity.

    INTRODUCTION
Top
Abstract
Introduction
References

Despite the impressive ability of natural enzymes to catalyze a broad array of reactions and utilize diverse substrates, attempts to make even minor modifications in enzyme activity or substrate specificity have proven to be difficult. Efforts to change the properties of existing enzymes (1-4) have highlighted a limitation to enzyme design; in most cases, we can consider only amino acid residues that constitute the active site. Enzymes, however, exert their functions not only through the chemical properties of the side chains of the amino acid residues that contact substrates and cofactors. Residues distant from an active site may be important in holding the catalytic residues in their required orientations. Charge distribution throughout a whole enzyme molecule may facilitate substrate binding by electrostatically guiding the substrate into the active site. These ideas are consistent with the fact that enzymes are macromolecules composed of hundreds of amino acid residues. It has been difficult to demonstrate by protein engineering the importance of residues that are remote from the active site because the number of these residues is large and the contribution of each residue may be modest. It is also beyond our present understanding to predict a priori the effects of the mutations of remote residues on enzymatic activity through changes in the complex architecture of tertiary and/or quaternary structure. Although random mutagenesis and directed evolution have recently proven to be useful in addressing such problems in the rational redesign of enzymes (5-10), creating enzymes with the desired activity remains challenging.

Aspartate aminotransferase (AspAT)1 is a homodimeric enzyme and catalyzes amino group transfer between acidic amino acids, aspartate and glutamate, and their corresponding 2-oxo acids (11). Each subunit has a pyridoxal 5'-phosphate (PLP) molecule at the active center. The structure (12, 13) and reaction mechanism (14) of AspAT have been studied extensively. AspAT from Escherichia coli shows moderate activity for aromatic amino acids. The activity for beta -branched amino acids, valine and isoleucine, is barely detectable and even lower than that for basic amino acids (15). Several studies were previously done to increase the activity for basic or aromatic amino acids (16-18). These results imply that the substrate specificity of AspAT like many other enzymes cannot be easily manipulated by mutating one or a few active site residues.

To alter the substrate specificity of AspAT toward beta -branched amino acids, we established an experimental system based on directed evolution where mutant AspATs with higher activity for branched-chain substrates, especially valine and 2-oxovaline, evolve during successive rounds of selection (10). Briefly, the selection system uses an auxotrophic E. coli strain, which is deficient in the gene for branched-chain amino acid aminotransferase. The higher the activity of a plasmid-encoded mutant AspAT for 2-oxovaline is, the faster the auxotrophic E. coli carrying the plasmid grows on a selection plate. After five rounds of the selection, the catalytic efficiency (kcat/Km) of mutant AspATs for beta -branched substrates was increased about 105-fold. One of the mutant AspATs showing the highest activity (AV5A-7) was analyzed in detail (10). The mutant had 13 amino acid substitutions, but interestingly, only one of the mutated residues seemed to interact directly with the substrate based on the three-dimensional structure of the wild-type AspAT. To elucidate the effects of these mutations on substrate binding, we set out to determine the three-dimensional structure of the mutant AspAT complexed with a beta -branched substrate analog. The affinity of AV5A-7 for valine was, however, still too low (Km = 400 mM) to yield a crystal of such an enzyme-inhibitor complex.

Here we describe the further improvement in both the catalytic efficiency and the Km value of AV5A-7 for valine. The affinity of a new mutant AspAT for valine is high enough to allow crystallization of a complex between the mutant enzyme and a valine analog. The crystal structure shows that the mutations in residues distant from the active site cause significant changes in the higher order structure of the enzyme, which influence substrate and cofactor binding.

    MATERIALS AND METHODS

Directed Evolution-- An auxotrophic E. coli strain, YJ103 (Delta ilvE::kan), the selection medium, 5' and 3' primers used for the polymerase chain reaction amplification of the coding region of aspC, and the construction of the expression plasmid were reported previously (10). Briefly, the first round of selection was done as follows. The coding region of the aspC gene containing the three mutations, Ser139 right-arrow Gly, Asn142 right-arrow Thr, and Asn297 right-arrow Ser, was subjected to DNA shuffling (6, 9, 19), and the mutant genes were ligated downstream of the promoter of the tetracycline resistance gene of pBR322. After a 45-h incubation at 37 °C, 116 colonies from a library of 5.6 × 107 colonies were picked up, and a mixture of the plasmids was prepared. As for the second and third rounds of selection, the conditions were the same as those for the first round except the incubation time: the second round, a 40-h incubation, a library size of 7.9 × 107 colonies, and 136 colonies picked; the third round, a 28-h incubation, a library size of 2.8 × 107 colonies, and 48 colonies picked. Among the 48 clones, 9 clones exhibiting the highest activity for 2-oxovaline were chosen, and the coding regions of the aspC genes were sequenced.

Expression, Purification, and Activity Measurement of Mutant AspATs-- The coding region of the mutant AspATs was subcloned into pUC18. The mutant enzymes were expressed in E. coli TY103 (20), which is deficient in the AspAT gene, and purified as described (21). The activity for each substrate was measured at 25 °C by spectrophotometrically monitoring the single turnover reaction using an Applied Photophysics stopped-flow apparatus (model SX.17MV) as described (15). The buffer system was 50 mM Hepes, pH 8.0, containing 0.1 M KCl and 10 µM EDTA.

Crystallography-- The crystals of ATB17 complexed with isovalerate were grown by the sitting drop vapor diffusion method. Three microliter drops containing 37 mg/ml protein were mixed with 1 µl of 0.2 M sodium isovalerate and 3 µl of the reservoir solution containing 1.6 M ammonium sulfate and 0.1 M Na·Hepes, pH 7.5. The drops were equilibrated against 0.5 ml of the reservoir solution at 20 °C. An x-ray data set was collected with a Rigaku R-AXIS IIc image plate detector mounted on a Rigaku RU-200 rotating anode generator operated at 40 kV and 100 mA with monochromatized CuKalpha radiation at room temperature. The oscillation images were processed and reduced using a data processing software, Rigaku PROCESS (22). Refinement of the structure began with the structure of the wild-type AspAT2 (12) as an initial model using X-PLOR 3.851 (23) with parameters derived by Engh and Huber (24). After conventional positional refinement, simulated annealing using the slow cool protocol was performed. The models were improved by conventional positional refinement and the isotropic B-factor refinement, and manual rebuilding using Xfit (25) on the omit map was calculated with the coefficients |Fo- |Fc|. After R-factors were adequately lowered, water molecules were added to the models, and the structures were further refined.

    RESULTS AND DISCUSSION

Creation of a Mutant AspAT with Higher Affinity for Valine-- Sequence analysis of the mutant AspATs obtained from the previous selection showed that 5 of the 13 substitutions found in AV5A-7, Asn34 right-arrow Asp, Ile37 right-arrow Met, Ser139 right-arrow Gly, Asn142 right-arrow Thr, and Asn297 right-arrow Ser, were conserved among all of the mutants examined, and these substitutions were found to be functionally important (10). The fact that one substitution (Val387 right-arrow Leu) that significantly increased the activity for beta -branched substrates was unique to AV5A-7 (10), however, implied that the sequence space had not yet been fully searched. Despite this finding, the initial selection system appeared to have reached its limit after five rounds of selection, because further increases in the catalytic efficiency for beta -branched substrates did not benefit the growth of the host E. coli cells under the most stringent selection conditions.

In the present study, we used two different strategies to find additional beneficial mutations. In one approach, we added each of the 20 unique substitutions found in four selected mutant AspATs, AV5A-1, AV5B-1, AV5B-4, and AV5B-5 (10), to AV5A-7 one mutation at a time and assayed each new mutant for 2-oxovaline activity (data not shown). Two substitutions, Ser361 right-arrow Phe and Ser363 right-arrow Gly, were chosen in this manner. In a second strategy, directed evolution was once again employed. This time, however, the experiment was started from a mutant AspAT that had three substitutions, Ser139 right-arrow Gly, Asn142 right-arrow Thr, and Asn297 right-arrow Ser, to facilitate the evolution. After three rounds of selection, mutant AspATs that showed similar activity for 2-oxovaline to that of AV5A-7 were obtained, and the coding regions of 9 mutant AspATs were sequenced. Each mutant AspAT had 4-9 additional substitutions, and, again, Asn34 right-arrow Asp and Ile37 right-arrow Met were conserved in all the mutants. After adding each potential substitution one at a time to AV5A-7/Ser361 right-arrow Phe/Ser363 right-arrow Gly, 3 substitutions, Ala11 right-arrow Thr, Phe24 right-arrow Leu, and Ile353 right-arrow Thr, were chosen. One of the 13 substitutions of AV5A-7, Glu7 right-arrow Val, was mutated back to the wild-type sequence because the substitution was found to decrease the expression level of AspAT while not affecting the activity for beta -branched substrates. The resulting mutant, ATB17, thus has 17 substitutions (Fig. 1A), 11 of which are clearly functionally important. As for the other 6 substitutions, Lys41 right-arrow Asn, Lys126 right-arrow Arg, Ala269 right-arrow Thr, Ala293 right-arrow Val, Ser311 right-arrow Gly, and Met397 right-arrow Leu, of which the contribution to the total effect was 10-20% in AV5A-7, the importance of each substitution could not be determined (10).


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Fig. 1.   Analysis of protein sequences and absorption spectra of AspATs. A, amino acid residues mutated in AV5A-7 (10) and ATB17. B, absorption spectra of the wild-type AspAT (solid line), AV5A-7 (dotted line), and ATB17 (broken line). All the spectra were measured in a 50 mM Pipes buffer, pH 6.5, at a protein concentration of 19 µM.

Characterization of the Mutant AspAT-- Compared with AV5A-7, the kcat/Km values of ATB17 for branched-chain substrates are increased, whereas those for acidic 2-oxo acid substrates are decreased (Table I). In particular, the Km value for valine is decreased 76-fold to 5.5 mM. The kcat/Km value of ATB17 for valine or 2-oxovaline is increased >2.1 × 106 or 6.7 × 105-fold, respectively, and that for isoleucine or 2-oxoisoleucine is increased >6.0 × 104 or 5.4 × 105-fold, respectively, compared with that of the wild-type AspAT. ATB17 retains significant activity for acidic substrates. Although this activity could have been easily eliminated by a single mutation at Arg292 (see below and also Refs. 16 and 17), none of the mutants examined had such mutations. Probably Arg292 was maintained simply because no selection pressure was applied to minimize the activity for acidic substrates or because acidic amino acids may serve as the amino group donors to 2-oxovaline in E. coli cells growing under the selection conditions.

                              
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Table I
Kinetic parameters of AspATs for branched and acidic substrates
Standard deviations are in parentheses.

During the purification of ATB17, we noticed that the color of the fractions containing the enzyme was orange, rather than yellow like the wild-type AspAT and AV5A-7. Thus, the absorption spectra of the purified AspATs were measured (Fig. 1B). AspAT has absorption bands in the region 300-500 nm, which derive from the bound PLP molecule and are influenced significantly by amino acid residues interacting with PLP (11, 26, 27). The wild-type AspAT and AV5A-7 exhibit two major bands around 360 and 430 nm, whereas in ATB17 the latter band is red-shifted to 450 nm and has a broad shoulder above 500 nm. The differences in the relative intensity of the two bands between the wild-type and mutant AspATs show that the pKa of the imine nitrogen of the Schiff base formed between PLP and Lys258 (26, 27) is decreased in both mutant AspATs. These changes in the absorption spectra imply that the electronic distribution within the pi -electron system of the bound PLP molecule is changed significantly by the mutations, although the nature of these changes is difficult to predict.

Despite such drastic changes in the substrate specificity and absorption spectrum, only one of the mutated residues in ATB17 appears to be located at a position contacting the substrate, and none of them interact directly with PLP. The higher affinity of ATB17 for valine allowed us to crystallize ATB17 in the presence of an amino-free valine analog, isovalerate, to further investigate the effects of the mutations.

Effects of the Mutations on the Tertiary and Quaternary Structure of AspAT-- The x-ray crystal structure of ATB17 complexed with isovalerate was solved at 2.4-Å resolution (Table II) and was compared with the wild-type AspAT complexed with an aspartate analog, maleate (Fig. 2). Three features stand out in the gross structure of ATB17. First, the spatial arrangement of the two subunits of the dimer is altered. Second, the domain motion may be enhanced in ATB17. Third, two clusters of mutated residues are observed, one shown in green (Leu-24, Asp-34, Met-37, and Asn-41) and the other shown in red (Thr-353, Phe-361, Gly-363, Leu-387, and Leu-397) in Fig. 2. These findings may provide clues to elucidate how the non-active site mutations influence the substrate binding of AspAT and thus are further described below.

                              
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Table II
Crystallographic parameters


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Fig. 2.   Superimposition of the ATB17-isovalerate complex (pink, large domain; gray, small domain) and the wild-type AspAT-maleate complex (purple). The backbone of one subunit of ATB17, of which the large domain (residues 49-325) was superimposed on that of the wild-type AspAT (31), is indicated by a thick line. The NH2- and COOH termini of the subunit are indicated (N and C). The side chains of the residues that were mutated in ATB17 are shown as follows: the two clusters of the residues (see text) are in red and green, and the other residues are in light blue. The coenzyme, pyridoxal 5'-phosphate (yellow), and the bound valine analog, isovalerate (dark blue), are also shown. Maleate of the wild-type AspAT-maleate complex is omitted. Figs. 2 and 4 were produced with MOLSCRIPT and RASTER 3D (32-34).

The rearrangement of the two subunits in ATB17 is evident. When the large domain (residues 49-325) of one subunit of ATB17 was superimposed on that of the wild-type AspAT, the other subunits of the two enzymes overlapped poorly (deviated about 1 Å in the core region of the large domain) (Fig. 2). This difference in the subunit arrangement likely affects substrate binding because the active site is located at the subunit interface, although it is not known which mutations caused this subunit rearrangement.

The structure of AspAT changes from an "open" to a "closed" conformation when the substrate binds to the enzyme (12, 13). In the ATB17-isovalerate structure, the domain closure is enhanced. The small domain of ATB17 comes closer to the active site compared with the corresponding domain of the wild-type AspAT. One of the clusters (Fig. 2, green), of which Met-37 is the only residue interacting with isovalerate, is located at the lid of the active site which closes against the bound substrate. The other cluster (Fig. 2, red) is in the core of the small domain, except Leu-397, and may affect the domain motion given that these residues are located at the domain interface. Thus, these two clusters may influence in concert the domain motion.

Detailed analysis may help us to understand how the two clusters of mutated residues cause the observed conformational changes. Fig. 3 shows that the enzyme structure is changed especially around the two clusters. The Leu20-Ile33 loop (the lid of the active site) shifts toward the active site while maintaining the overall configuration of the loop structure (Fig. 4B). The average deviation is about 1.5 Å, but the deviation is larger around the tip of the loop than around its bottom. This cluster of mutations is located at the hinge-like region and thus may cause the shift of the loop. On the other hand, the largest deviation in the whole molecule is observed at the domain interface (Fig. 3, position 363, and Fig. 4C). The backbone of Gly363-Gly364 has flipped toward the solvent side. The Ser361 right-arrow Phe substitution would cause a steric hindrance due to its phenyl side chain. The Ser363 right-arrow Gly substitution yields two consecutive glycine residues of which the flexible backbone would flip to release the unfavorable strain. The effects of these two substitutions are, however, not fully cooperative because each substitution independently increased the activity for 2-oxovaline when added to AV5A-7. Other changes observed in Fig. 4C would have occurred to readjust the packing of the side chains surrounding the cluster of the mutated residues. All the above findings, including changes in the dimer interface, likely influence the substrate specificity of AspAT, but we cannot explain how these changes allow AspAT to accommodate beta -branched substrates.


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Fig. 3.   Deviations of the Calpha atoms between the ATB17-isovalerate complex and the wild-type AspAT-maleate complex. The large domains of the two structures (269 Calpha atoms for each) were superimposed with the root mean square deviation of 0.316 Å. The root mean square deviation for all the 396 Calpha atoms was 0.552 Å. Figs. 2 and 4 show the superimposed images obtained in this manner. The numbers of the residues which showed large deviations are indicated.


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Fig. 4.   Close-up views of the active site and the two clusters of the mutated residues. The residues are colored as shown in Fig. 2. The structure of the ATB17-isovalerate complex is indicated by thick gray lines and that of the wild-type AspAT-maleate complex is indicated by thin purple lines. A, several water molecules are introduced into the active site of ATB17 (light blue spheres). One water molecule (WAT1) is located at almost the same position as a water molecule observed in the wild-type AspAT (a purple sphere). Arg292, Val293, and Ser297 belong to the other subunit of the dimer (asterisks). B and C, viewed from the same direction as in Fig. 2.

The Active Sites and Substrate-binding Modes of the Mutant and Wild-type AspATs-- Trp140 is thought to adjust the tilt of the PLP molecule during the course of catalysis by AspAT (11, 14). In the ATB17-isovalerate structure, large conformational changes are observed in the Pro138-Thr142 loop (Fig. 3 and Fig. 4A). This loop contains two substitutions, Ser139 right-arrow Gly and Asn142 right-arrow Thr. Trp140 is moved toward the "bottom" of the active site, making more space in the substrate binding pocket. The indole ring of Trp140 is stacked against the pyridine ring of PLP by the hydroxyl group of Thr142, which is bulkier than the hydrogen atom at the corresponding position of the side chain of the original Asn142. The resulting tight interaction of the pi  election systems of the indole ring and PLP may in part explain the spectral changes observed in ATB17. Interestingly, one of the evolved mutants we reported previously (10), AV5B-4, had an isoleucine residue at position 142, which also has a bulky methyl group at Cbeta of the side chain. Thus, the PLP molecule may be required to be pushed back by Trp140 for AspAT to bind beta -branched substrates.

The wild-type AspAT has two arginine residues, Arg292 and Arg386, that are essential for substrate binding. Arg292 interacts with the side chain carboxylate group of acidic substrates. The side chain of Arg292 of ATB17 protrudes into solvent, whereas that of the wild-type AspAT flips toward the active site interacting with the carboxylate group of maleate (Fig. 4A). The same shift in the position of Arg292 was observed previously in the crystal structure of a mutant AspAT complexed with aromatic substrate analogs (28). It is therefore possible that the difference in the position of Arg292 between the two enzymes is caused by the difference in the bound substrate analogs, dicarboxylic maleate and monocarboxylic isovalerate, rather than by the mutations of ATB17. Arg386 is also an important residue of which the side chain interacts with the alpha -carboxylate group of all amino acid substrates. The orientation of the Arg386 side chain of ATB17 remains essentially unaltered despite the large conformational changes adjacent to Arg386 (Fig. 4, A and C).

One of the side walls of the active site consists of the residues belonging to the other subunit (Fig. 4A, asterisks). Thus, the changes in the subunit interface in ATB17 have caused deviations of these residues. This portion of the active site also contains two substitutions, Ala293 right-arrow Val and Asn297 right-arrow Ser. Although the functional importance of the Ala293 right-arrow Val substitution is not clear, the Asn297 right-arrow Ser substitution increased the activity of AV5A-7 for 2-oxovaline (10). It was reported previously for the mutant AspAT with increased activity for aromatic substrates that the Asn297 right-arrow Ser substitution eliminates a structural water molecule which is held in place by the side chains of Ser296 and Asn297 in the wild-type AspAT, concomitantly introducing new water molecules (28). Malashkevich et al. (28) proposed that this substitution makes space for the binding of bulky aromatic substrates. In our case, however, the side chain of isovalerate is much smaller than those of aromatic substrates, and, indeed, a water molecule (Fig. 4A, WAT1) is re-introduced at almost the same position, constituting the side wall of the isovalerate-binding pocket together with the other newly introduced water molecules. We thus cannot explain how the Asn297 right-arrow Ser substitution increases the activity for valine.

Met37 constitutes the upper wall of the isovalerate binding pocket and is the only mutated residue contacting isovalerate. The Ile37 right-arrow Met substitution would apparently benefit the binding of beta -branched substrates by removing the steric hindrance between the gamma -methyl groups of the Ile37 side chain and the bound substrate. To determine how important this substitution is, we mutated Met37 of ATB17 back to isoleucine. The kcat/Km values of this revertant for valine and 2-oxovaline were decreased 4- and 2-fold, respectively; the kcat, Km, and kcat/Km values for valine were 15 ± 1 s-1, 30 ± 5 mM, and 500 s-1 M-1, respectively, and those for 2-oxovaline were 31 ± 2 s-1, 1.8 ± 0.3 mM, and 1.7 × 104 s-1 M-1, respectively. This shows that the Ile37 right-arrow Met substitution contributes only 5-10% to the total effect of all the mutations of ATB17 (calculated as follows: (1 - (RT ln{(kcat/Km)ATB17/Met37 right-arrow  Ile/(kcat/Km)WT})/(RT ln{(kcat/Km)ATB17/(kcat/Km)WT})) × 100 (%)).

Conclusions-- The isolation of a mutant AspAT with a million-fold increase in the catalytic efficiency for a non-native substrate shows that directed evolution is a powerful technique for altering enzyme activity. Our present selection system could also be used to produce an enzyme with transaminase activity from that with an unrelated activity. The three-dimensional structure of a complex between the mutant AspAT and a substrate analog suggests that conformational changes in the enzyme are responsible for the alteration in the substrate specificity. Significantly, only one of the 17 mutated residues contacts the substrate directly. Several of the mutations that particularly enhance the activity for beta -branched substrates are located >10 Å distance from the active site.

Most attempts to redesign enzyme activity by mutating only active site residues have met with limited success. It may turn out to be common rather than exceptional that changing enzyme activity requires remodeling the active site through changes in the backbone flexibility, domain motion, or subunit arrangement. Results supporting this idea were recently reported for a catalytic antibody (29, 30). These findings emphasize the benefits of directed evolution over rational design and would justify present and future intense efforts to develop new strategies for directed evolution.

    ACKNOWLEDGEMENTS

We thank K. Hirotsu for help in the x-ray data collection and V. W. Cornish and D. R. Liu for helpful comments on the manuscript.

    FOOTNOTES

* This work was supported by the Ministry of Education, Science, Sports, and Culture of Japan (to T. Y.) and the Japan Society for the Promotion of Science ("Research for the Future" Program) (to H. K.).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 structure factors (code 1yoo) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.

Dagger To whom correspondence should be addressed: Dept. of Biochemistry, Osaka Medical College, Takatsuki, Osaka 569-8686, Japan. Tel.: 81-726-83-1221 (ext. 2644); Fax: 81-726-84-6516; E-mail: med001{at}art.osaka-med.ac.jp.

The abbreviations used are: AspAT, Aspartate aminotransferase; PLP, pyridoxal 5'-phosphate; 2-oxovaline, 2-ketoisovaleric acid; Pipes, 1,4-piperazinediethanesulfonic acid.

2 The atomic coordinates for the crystal structure of the wild-type AspAT have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY, code 1ART (12).

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