First published online July 22, 2005; 10.1104/pp.105.062885
Plant Physiology 138:2260-2268 (2005)
© 2005 American Society of Plant Biologists
BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES
Structure-Based in Vitro Engineering of the Anthranilate Synthase, a Metabolic Key Enzyme in the Plant Tryptophan Pathway1,[w]
Takuya Kanno,
Akira Komatsu,
Koji Kasai,
Joseph G. Dubouzet,
Minako Sakurai,
Yasuko Ikejiri-Kanno,
Kyo Wakasa and
Yuzuru Tozawa*
Cell-Free Science and Technology Research Center (T.K., Y.I.-K., Y.T.), and Venture Business Laboratory, Ehime University, Matsuyama 790–8577, Japan (Y.T.); National Institute of Crop Science, Tsukuba 305–8518, Japan (A.K., K.W.); Japan Science and Technology Agency for Core Research for Evolutional Science and Technology Plant Functions and Their Control (T.K., A.K., J.G.D., K.K., M.S., K.W., Y.T.); and Mitsubishi Kagaku Institute of Life Sciences, Yokohama Research Center, Yokohama 227–8502, Japan (T.K., Y.T.)
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ABSTRACT
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Rice (Oryza sativa) anthranilate synthase  -subunit, OASA2, was modified by in vitro mutagenesis based on structural information from bacterial homologs. Twenty-four amino acid residues, predicted as putative tryptophan binding sites or their proximal regions in the OASA2 sequence, were selected and 36 mutant OASA2 genes were constructed by PCR-based site-directed mutagenesis. Corresponding mutant proteins were synthesized in a combination of two in vitro systems, transcription with a bacteriophage SP6 RNA polymerase and translation with a wheat-embryo cell-free system. Enzymatic functions of the mutant proteins were simultaneously examined, and we found six mutants with elevated catalytic activity and five mutants with enhanced tolerance to feedback inhibition by tryptophan. Moreover, we observed that some sets of specific combinations of the novel mutations additively conferred both characteristics to the mutant enzymes. The functions of the mutant enzymes were confirmed in vivo. The free tryptophan content of mutant rice calli expressing OASA2 enzyme with a double mutation was 30-fold of that of untransformed calli. Thus, our in vitro approach utilizing structural information of bacterial homologs is a potent technique to generate designer enzymes with predefined functions.
Anthranilate synthase (AS) is a metabolic key enzyme of the Trp synthetic pathway in plants and microbes, and its activity is regulated by feedback inhibition by Trp. The AS enzyme consists of two subunits, and  ; the -subunit possesses a catalytic domain for the synthesis of anthranilate from chorismate and a regulatory domain for Trp feedback inhibition. To date, three-dimensional structures of several bacterial AS enzymes have been solved by x-ray crystallography. The molecular mechanism for feedback inhibition of AS activity by Trp has been clarified (Knöchel et al., 1999 ; Morollo and Eck, 2001; Spraggon et al., 2001). Some of the residues assigned as the Trp-interacting site have also been characterized genetically by the mutation analysis in various microbes and in several plants (Matsui et al., 1987; Caligiuri and Bauerle, 1991; Graf et al., 1993; Kreps et al., 1996; Li and Last, 1996; Song et al., 1998). In fact, some of the feedback-insensitive mutant enzymes found in these species have been utilized for generating high Trp producing microbes or plants (Cho et al., 2000; Tozawa et al., 2001; Zhang et al., 2001; Yamada et al., 2004). In addition, naturally occurring feedback-insensitive AS enzymes have been cloned and characterized from Ruta graveolens (Bohlmann et al., 1995, 1996) and tobacco (Nicotiana tabacum; Song et al., 1998).
We have previously identified two isozymes for both - and -subunits, and have revealed the distinct functional properties of two AS -subunit isozymes, OASA1 and OASA2, in rice (Kanno et al., 2004). However, OASA2 showed inferior characteristics to OASA1 in terms of both feedback insensitivity and catalytic activity; nevertheless, OASA2 is more abundantly expressed than OASA1 in the rice plant (Kanno et al., 2004). In this study, we used bacterial enzyme structure to identify probable Trp-interacting amino acid residues and their flanking regions and performed site-directed mutagenesis on these sites to investigate the mutation effects on catalytic activity and feedback inhibition.
Utilization of structural information from homologous proteins to design novel enzymes is one of the practical applications of structural biology. Structure-based protein engineering is a more reasonable strategy compared to general random mutagenesis. We employed PCR-mediated in vitro site-directed mutagenesis in combination with wheat-embryo cell-free protein synthesis to establish a high-throughput system. Here we describe (1) identification of novel mutations in OASA2 that modulate enzymatic functions; (2) combinations of mutations yielding improved distinct properties to OASA2; (3) kinetic studies and speculations about the effects of these mutations on the enzymatic functions, from the viewpoint of protein structure; and (4) in vivo performance of the mutant OASA2s in transgenic rice calli and in yeast (Saccharomyces cerevisiae) transformants.
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RESULTS
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Mutation Analysis of OASA2
To identify the amino acid residues that potentially modulate the functions of the OASA2-enzyme, the AS -subunit protein sequences from various species were aligned with OASA2 (Fig. 1B). The genetic and protein structural studies on bacterial AS revealed several amino acid residues that function in binding the free Trp molecule during the feedback inhibition (Matsui et al., 1987; Caligiuri and Bauerle, 1991; Graf et al., 1993; Morollo and Eck, 2001; Spraggon et al., 2001). These residues are dispersed within at least three regions in the protein primary structure (Fig. 1A). Among these amino acids, OASA2 Glu-125, Ser-126, Pro-366, Tyr-367, Met-368, and Gly-521, which correspond to Glu-39, Ser-40, Pro-291, Tyr-292, Met-293, and Gly-454 of the Salmonella typhimurium TrpE (SwissProt accession no. P00898), are all conserved in the aligned sequences (Fig. 1B). This complete conservation of several amino acids among the family members agrees well with the fact that these residues had been identified as part of a Trp-interacting site in crystallographical studies (Morollo and Eck, 2001; Spraggon et al., 2001). The other potential Trp-interacting amino acid residues in S. typhimurium TrpE, such as Leu-38 and Cys-465, were variable in the sequences of other family members. These variations indicate that some substitutions in these residues allow the enzymes to retain a common function. On the other hand, in the three potential Trp-interacting regions, OASA2 Tyr-349, Asn-351, Val-371, Leu-520, and Leu-530 uniquely differ from the corresponding residues in the other plant enzyme sequences (Fig. 1B). Based on this information, the putative Trp-interacting amino acid residues, the flanking residues of these potential Trp-interacting sites in OASA2, the residue corresponding to the chorismate binding site, and the residues showing unique variations in only the OASA2 sequence, were selected for mutation analysis in this study. The 24 residues thus chosen are summarized in Supplemental Table I. Amino acid substitutions by site-directed mutagenesis were carried out by the PCR method, as described in "Materials and Methods." Oligonucleotide primers, used for the mutagenesis and the template preparation by PCR, are listed in Supplemental Table II. The prepared DNA templates were used for in vitro mRNA synthesis, and then the mRNAs were subjected to in vitro protein synthesis. The AS activity of each synthesized protein was assayed as previously described (Kanno et al., 2004).
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Figure 1. Sites of the amino acid substitutions in OASA2. A, Functional domains in the OASA2 polypeptide. cTP indicates the chloroplast transit peptide in the amino terminus. I, II, and III indicate regions including putative binding sites of the free Trp moiety, predicted from structural studies on bacterial AS enzymes (Morollo and Eck, 2001; Spraggon et al., 2001). Dots indicate putative chorismate binding sites predicted from structural studies (Morollo and Eck, 2001; Spraggon et al., 2001). B, Alignment of amino acid sequences corresponding to regions I, II, and III of the AS -subunit family proteins. The sequences shown are: OASA1 and OASA2, rice AS -subunits OASA1 and OASA2 (GenBank accession nos. AB022602 and AB022603); ASA1 and ASA2, Arabidopsis ASA1 (M92353) and ASA2 (M92354); RASA1 and RASA2, Ruta graveolens AS1 (L34344) and AS2 (L34343); TASA2, tobacco ASA2 (AF079168); Ss-TrpE, S. solfataricus TrpE (Q06128); St-TrpE, S. typhimurium TrpE (P00898); and Sm-TrpE, S. marcescens TrpE (AAA57308. Hyphens indicate gaps introduced to optimize the alignment. Identical or similar residues among the various proteins are indicated by dark or light shading, respectively, and amino residue numbers are shown on both ends. Arrowheads mark amino acids of OASA2 mutated in this study. H, , and B indicate -helices, -strands, and -hairpins, respectively.
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The first round of mutation analysis was done for 36 various mutations. An ammonium-dependent AS assay was performed at three different concentrations of Trp (0 µM, 10 µM, and 100 µM) for each single AS -subunit enzyme. This mutation-scan revealed several mutation points that affected the enzymatic activity or the Trp-feedback inhibition of the enzyme (Fig. 2). Mutations that enhanced the catalytic activity were Y349A, Y349F, N351D, A380S, L520F, and L530D. In contrast, mutations such as N363A, N363D, P364A, P364L, P366A, G518A, and A532Y abolished the enzymatic activity. The Y516A mutation in the chorismate binding catalytic site (Spraggon et al., 2001) also produced a nonfunctional protein (data not shown). The mutations that conferred Trp-insensitive activity were S126F, S365A, Y367A, A369L, and G521A (Fig. 2). In contrast, N351A, G522A, and G522Y increased the sensitivity to the Trp feedback inhibition (Fig. 2).
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Figure 2. Ammonium-dependent AS activity of the synthesized OASA2 mutant proteins. The AS activities of the OASA2 derivatives, carrying single mutation, synthesized in the translation reaction mixture were determined in the presence of 100 mM NH4Cl. The assays were performed independently in the presence (10 µM or 100 µM) or absence of Trp. Letters under the graph indicate the mutant OASA2 proteins generated by single amino acid substitutions (for example, E125A means the substitution of the Glu residue with Ala at amino acid no. 125) or the wild-type protein (wt). The OASA2 proteins in the reaction mixture were quantified by immunoblotting, as described previously (Kanno et al., 2004). The specific activity was calculated based on the amount of OASA2 protein in the reaction mixture. Bars indicate SDS (n = 3).
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Combination of the Mutations in OASA2
The single-mutation analysis identified five feedback-insensitive mutations (Fig. 2). However, except for A369L, they reduced enzyme catalytic activities (Fig. 2). We added another mutation to improve activity of these feedback-insensitive single mutant enzymes. Among these, the addition of the L530D mutation exhibited positive effects on the enzyme catalytic activity, except in the G521 background (Fig. 3, A–E). Particularly, the combination of Y367A and L530D prominently improved the activity to 8-fold of that of the single mutant Y367A (Fig. 3C). On the other hand, the performance of the A369L mutant enzyme was not improved by additional mutations (Fig. 3D). As for the G521 mutant, there was no positive effect in combinations with other mutations (Fig. 3E).
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Figure 3. Combined effects of mutations on the enzymatic functions. The second mutation was introduced in the feedback-insensitive OASA2 derivatives S126F (A), S365A (B), Y367A (C), A369L (D), and G521A (E). Enzymatic functions of the engineered proteins were examined as described in the legend of Figure 2.
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Kinetic Analysis of Mutant AS Enzymes
Single- and double-mutation analyses were carried out based on the assay of the OASA2 single-subunit enzyme. The AS enzyme requires the -subunit to acquire more stable and enhanced enzymatic functions, including the Gln-dependent activity (Kanno et al., 2004). We performed enzyme assays in vitro to examine the enzymatic functions of the reconstituted OASA2 derivatives with the -subunit, OASB1. The functions of the reconstituted enzymes were characterized as mixtures of purified OASA2 derivatives and unpurified OASB1. The reaction mixture containing the OASA2 derivative protein was reconstituted with that including OASB1, and the enzyme assay was carried out as previously described (Supplemental Fig. 2; Kanno et al., 2004). The kinetic parameters of the reconstituted AS derivatives are shown in Table I. Similar to the results from the single-subunit enzyme assay (Fig. 3), double mutants such as S126F/L530D and Y367A/L530D showed improved catalytic activities as compared to single mutants, S126F, Y367A, or the wild type (Supplemental Fig. 2). On the other hand, the A369L single mutant enzyme showed less activity than expected from the results of the screening assay (Fig. 3). Interestingly, A369L/L530D achieved higher catalytic activity than A369L in the reconstituted enzyme (Table I; Supplemental Fig. 2A), in contrast to the results obtained from the single-subunit enzyme assay (Fig. 3D). These observations indicated that the addition of the L530D mutation in S126F, Y367A, and A369L effectively improved the enzyme catalytic function in the assembled form of the AS enzyme.
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Table I. Kinetic parameters of Gln-dependent AS activity of the wild-type and mutants of OASA2, reconstituted with OASB1
Parameters were determined for the OASA2 N49 wild-type protein or its mutant derivatives reconstituted with OASB1N58 and are means from at least three independent experiments.
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Next, the sensitivities of the assembled enzymes against Trp feedback inhibition were examined with various concentrations of Trp (Supplemental Fig. 2B). The calculated IC50 values of these enzymes are listed in Table I. As shown in Table I and Supplemental Figure 2, the mutant enzymes, S126F/L530D and Y367A/L530D, exhibited significantly higher insensitivities to high concentrations of Trp. The more than 80-fold increase of the IC50 of S126F/L530D and 16-fold increase of Y367A/L530D compared with the OASA2 wild-type enzyme demonstrate the remarkable improvement of the enzyme function by the introduction of the S126F/L530D or Y367A/L530D double mutation into OASA2.
Functional Analysis of Mutant OASA2 Genes in Vivo
To confirm and characterize the in vivo functions of the mutant OASA2 proteins, a TRP2 deficient yeast strain was transformed with the GAL4 promoter-driven OASA2-derivative expressing plasmid. We analyzed the phenotype and the free Trp content in the cells. All of the OASA2 mutant genes could complement the functional loss of TRP2, which is the yeast homolog of the AS -subunit, even in normal synthetic medium without NH4Cl, indicating the sufficient utilization of TRP3, the endogenous yeast AS -subunit, in the Gln-dependent AS enzyme (data not shown). A quantitative analysis of the soluble Trp in these yeast transformants revealed that some of the mutant OASA2 transformants obviously accumulated higher levels of Trp than the wild-type OASA2 transformant (Table II). The Y367A/L530D expressing yeast strain achieved the highest accumulation of free Trp in the cells, 2.3-fold of that of cells expressing wild-type OASA2. In contrast, the transformant expressing S126F/L530D did not show remarkably high Trp accumulation, which was expected from the kinetic data of reconstituted form with rice AS -subunit (Table I; Supplemental Fig. 2). The results demonstrate that, in combination with the yeast AS -subunit, the mutant OASA2 proteins improved the cellular Trp concentration due to their insensitivity to Trp.
To clarify the in vivo function of the mutant OASA2 genes, rice cells were transformed with constructs for expressing OASA2 wild type, OASA2 (Y367A), or OASA2 (Y367A/L530D). Transformed calli were selected on media containing hygromycin, and transgene expression was confirmed by RNA gel-blot hybridization (Fig. 4). Free Trp concentrations of these calli lines are shown in Table III. The free Trp content in calli expressing OASA2 (Y367A) or OASA2 (Y367A/L530D) was 176 to 532 nmol or 1,106 to 1,243 nmol per milligram fresh weight calli, which is 5.5- to 16.6-fold or 34.6- to 38.8-fold of that in nontransformed calli, respectively. The results demonstrate a drastic increase in Trp by a combination of amino acid substitutions in the mutant enzyme.
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Figure 4. RNA gel-blot analysis of rice calli expressing OASA2 derivatives. Top, Total RNA (5 µg) was subjected to RNA gel-blot analysis with a digoxigenin-labeled OASA2 riboprobe. Bottom, The ethidium bromide-stained agarose gel is shown. NB, Untransformed Nipponbare calli; lanes 1 to 4, transformants expressing the wild-type OASA2 transgene (transformant line nos. WT13, WT22, WT28, and WT51, respectively); lanes 5 to 8, calli expressing OASA2(Y367A) transgene (line nos. Y1, Y9, Y29, and Y50, respectively); and calli expressing OASA2(Y367A/L530D) transgene (line nos. YL50, YL65, YL66, and YL68, respectively). Arrow indicates the transgene-derived transcripts.
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Table III. Free Trp content in transformed rice calli
The names of control and transformed lines correspond to those described in the legends of Figure 4.
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DISCUSSION
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In this study, we performed a semicomprehensive mutational analysis of putative Trp-binding regions and their franking regions in the OASA2 open reading frame (ORF; Fig. 1B; Supplemental Table I). This analysis revealed six amino acid substitutions that improved the catalytic activity, five substitutions that elevated the insensitivity to Trp feedback inhibition, three substitutions that enhanced the sensitivity to Trp (Fig. 2), and eight substitutions that abolished the catalytic activity (data not shown). Interestingly, many of the mutated residues that improved catalytic activity, such as Tyr-349, Asn-351, Leu-520, and Leu-530, are uniquely different from their counterparts in other plants in the sequence alignment (Fig. 1B). Substitution of OASA2 residues with residues conserved in other homologs improved the activity of the OASA2 enzyme (Fig. 2). Rice has two AS -subunit genes, OASA1 and OASA2 (Tozawa et al., 2001); the dominantly expressed OASA2 enzyme shows inferior function as compared with the less abundant OASA1 enzyme, in terms of the catalytic activity and the Trp feedback insensitivity (Kanno et al., 2004). We previously suggested that the OASA2 enzyme regulates the Trp level in the cell (Kanno et al., 2004). In support of this hypothesis, transgenic rice cells expressing a higher level of the wild-type OASA2 than untransformed cells had a lower cellular content of Trp (0.2- to 0.6-fold) compared with untransformed cells (Table III). It is also probable that rice has acquired a less efficient Trp biosynthesis system due to changes in the functions of OASA2 during its evolution. The energetic cost of Trp synthesis is considered to be the highest among the synthesis of all amino acids (Hrazdina and Jensen, 1992). Minimizing the energetic cost of the Trp pathway might be a reasonable strategy of energy conservation in rice.
In contrast to rice enzymes, naturally occurring feedback-insensitive AS enzymes have been found in other plants such as R. graveolens and tobacco (Bohlmann et al., 1995, 1996; Song et al., 1998). It has been postulated that the elicitor-inducible AS1 takes significant roles in the secondary metabolism of R. graveolens (Bohlmann et al., 1996).
Five mutations that significantly improved feedback insensitivity (Fig. 2) are found in three different regions in the OASA2 sequence (Fig. 1A), indicating that these three regions in the OASA2 sequence are involved in Trp binding. As discussed above, these regions have also been associated with the binding of Trp to the bacterial enzymes (Knöchel et al., 1999; Morollo and Eck, 2001; Spraggon et al., 2001). Our results also clearly reflect a part of the results from genetic studies (Caligiuri and Bauerle, 1991). The mutations corresponding to OASA2 Ser-126 and Ala-369 were reported from the mutant analysis of AS -subunit of Brevibacterium lactofermentum (Matsui et al., 1987) and S. typhimurium (Caligiuri and Bauerle, 1991), respectively, while the other three mutations are novel. The Ala-369 residue varies among its counterparts (Fig. 1B), indicating that this residue can tolerate some variation without a loss of catalytic function. On the other hand, the other four mutated residues, Ser-126, Ser-365, Tyr-367, and Gly-521, are completely conserved in the alignment (Fig. 1B). The Ser-126 locates in the highly conserved amino acid motif, Leu-Leu-Glu-Ser, in the amino terminal end of the OASA2, and the previous reports described that mutation in the corresponding residue in the microorganisms and yeast affects feedback inhibition (Matsui et al., 1987; Caligiuri and Bauerle, 1991; Graf et al., 1993). Structural study on the S. typhimurium AS demonstrated that in the Ser of the Leu-Leu-Glu-Ser motif position at the Trp feedback regulatory site, Ser binds the Trp moiety by forming hydrogen bond between the Ser-40 O and the amino group of the free Trp (Morollo and Eck, 2001). Combining these with our results, we speculate that mutation of the Ser-126 of the OASA2 abolished affinity to the Trp moiety in this regulatory site, resulting in a loss of capability to form the inactive "T-state" that restricts the entry of substrate chorismate into the catalytic domain (Knöchel et al., 1999; Morollo and Eck, 2001). The Tyr-367 has also been assigned as one of the most significant residues that hold the bound Trp moiety in the feedback regulatory site (Morollo and Eck, 2001; Spraggon et al., 2001). The Ser-365 residue lies close to Tyr-367, and therefore its mutation may affect the polypeptide structure surrounding Tyr-367. As for Gly-521, Gly is a residue with only a hydrogen atom as a side chain so it can adopt a wider range of conformations than other residues (Richardson, 1981). Therefore, one of the possible effects of the G521A mutation is a restriction of the conformational changes of the polypeptide containing the substituted residue, Ala-521.
As demonstrated, more than one-half of the 36 mutations affected the enzyme catalytic activity (Fig. 2), indicating that the structures of the Trp-interacting regions in OASA2 significantly modulate the catalytic activity. Structural studies on bacterial AS enzymes revealed that the distance between the residues involved in catalysis and feedback regulation is approximately 18 Å in the Serratia marcescens AS (Spraggon et al., 2001) and 20 Å in the Sulfolobus solfataricus AS (Knöchel et al., 1999). These studies demonstrated that Trp binding to a regulatory site alters the conformation of the enzyme structure and prevents chorismate entry to the catalytic site (Knöchel et al., 1999; Morollo and Eck, 2001; Spraggon et al., 2001). Our results obtained from the OASA2 mutational analysis support the speculations drawn from structural studies on the bacterial AS enzyme, demonstrating the functional significance of the amino acid residues in the Trp-binding regions.
On the other hand, the effects of Asn at residue number 323 in the OASA1 mutant and 351 in OASA2 are different (Fig. 1B). In the case of OASA1, the D323N mutation in OASA1D conferred feedback-insensitivity to the enzyme (Tozawa et al., 2001). In contrast to OASA1 and OASA1D, the OASA2 wild-type enzyme has Asn at this position and shows highly sensitive activity to Trp (Kanno et al., 2004). The enzyme assay for the OASA2 mutants in this study shows that the feedback-sensitivity was altered by the N351A mutation, but not by N351D, and that the catalytic activity was increased by the N351D mutation, but not by N351A (Fig. 2). These results indicate that the amino acid at position 351 of OASA2 certainly affects enzyme functions. We conclude that the different effects of the mutations of OASA2 residue 351 and the corresponding OASA1 residue are due to differences in the structures of their protein backbones. In addition, it has been reported that R. graveolens and tobacco possess naturally occurring Trp feedback-insensitive AS -subunit, AS1 and ASA2, respectively (Bohlmann et al., 1996; Song et al., 1998). As shown in Figure 1B, these two AS -subunits have uniquely different amino acid sequences at positions 131 and 132 (position no. based on the OASA2 sequence). Although structural analyses for bacterial AS enzymes (Knöchel et al., 1999; Morollo and Eck, 2001; Spraggon et al., 2001) have not assigned corresponding amino acids to these as Trp-interaction site, there still may be other potential amino acid residues that can be targeted to alter the functions of the OASA2 protein.
Mutation of the AS enzyme to confer insensitivity to Trp inhibition is a major objective for engineering the Trp biosynthetic pathway (Tozawa et al., 2001). Of the four mutants, only A369L retained its original catalytic activity level, but the other three had reduced activities. We then combined another type of mutation, which enhanced the catalytic activity, with each feedback-insensitive mutation, and found that the addition of the L530D mutation to S126F, Y367A, and A369L remarkably improved the catalytic activity, without a loss of the feedback-insensitivity (Fig. 3). We have confirmed the characteristics of these mutant OASA2 proteins by means of a kinetic study of the AS enzymes reconstituted with the -subunit, OASB1 (Table I; Supplemental Fig. 2). The assay of the -AS enzymes showed significantly improved enzymatic functions than those observed in the single-subunit enzyme assay (Table I; Figs. 3). Each kinetic parameter clearly demonstrates the positive effect of introducing the L530D second mutation into S126F, Y367A, or A369L (Table I). It is noteworthy that the S126F/L530D, Y367A/L530D, and A369L/L530D double mutations also showed higher IC50 values by >80-, 16-, and 9-fold, respectively, of that of the original enzyme (Table I).
We have previously reported that the expression of the feedback-insensitive OASA1 mutant gene improved the Trp content in some important crops plants (Tozawa et al., 2001; Yamada et al., 2004). In addition to biochemical examinations, the in vivo functions of the mutant OASA2 genes have been demonstrated by transformation analyses in yeast (Table II) and rice (Table III). The expression of the novel OASA2 mutant enzymes improved the free Trp content in yeast cells (Table II) and in rice calli (Table III). A yeast mutant expressing the mutant OASA2 protein moderately increased the soluble Trp content (1.6–2.3-fold) compared to the cell expressing OASA2 wild-type enzyme (Table I). In the yeast strain lacking the AS -subunit (TRP2), Gln-dependent AS enzyme is presumably reconstituted with the OASA2-derivative and the endogenous AS -subunit (TRP3). The modest increment of the free Trp in yeast transformants is likely due to the reconstitution efficiency or the enzymatic properties of the reconstituted AS. In contrast, the Trp content in the rice calli expressing OASA2 (Y367A/L530D) was significantly increased up to 39-fold compared to the calli expressing OASA2 wild-type gene (Table II). The higher Trp accumulation in rice calli expressing the OASA2 (Y367A/L530D) mutant gene as compared to rice calli expressing the OASA2 (Y367A) mutant gene shows good agreement with the kinetic values (Table I; Vmax, kcat, and IC50) of each mutant enzyme, indicating the additive effect of the two mutations. Thus, these designer genes encoding mutant OASA2 protein have good potential for metabolic engineering of the Trp pathway in crops. For the other mutant OASA2 genes, such as S126F, A369L, S126F/L530D, and A369L/L530D, rice transformation analysis is currently in progress.
In this study, we found mutations that modified enzymatic properties, and obtained satisfactorily engineered mutant enzymes, OASA2 (S126F/L530D), OASA2 (Y367A/L530D), and OASA2 (A369L/L530D). Among them, it was confirmed that OASA2 (Y367A/L530D) is practically useful for molecular breeding of crops to improve free Trp content (Table III). As demonstrated, a site-directed mutation analysis aiming to confer novel function to the target protein requires some extent of comprehensiveness for selecting the different criteria for the points to be mutated in the amino acid sequence. In this regard, our biochemical system exhibited the high performance of the wheat-embryo cell-free protein synthesis system. The utilization of this system allows us to save time by skipping the bacterial recombination steps in the process. For instance, one is able to accomplish the procedure from the preparation of the DNA template by PCR to starting the in vitro translation within a day. Although it was a special case, we did not need to purify the synthesized product from the reaction mixture. Therefore, the whole process, from template DNA preparation to protein synthesis, was completed within 2 d by the all-PCR system, whereas the Kunkel mutagenesis method (Kunkel, 1985) requires more than a week, because the system included a bacterial recombination process. Thus, the cell-free mutation scanning system is a potent technology, which is applicable for the engineering of various kinds of functional proteins.
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MATERIALS AND METHODS
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Site-Directed Mutagenesis
Site-directed mutagenesis of the OASA2 gene was carried by in vitro overlap-extension PCR (Higuchi et al., 1988). Two separate PCRs were performed to amplify two halves of the OASA2 gene, using four primers. An outside-forward primer (OASA2-N49, 5'-cctcttccagggcccaATGTGCTCCGCGGGGAAGCC-3'; the underlined sequence is an artificially introduced initiation codon and the lowercase letters indicate the linker sequence for the split-primer method; Sawasaki et al., 2002) was paired with a middle-reverse mutation primer to generate the first half of the gene; an outside-reverse primer (the plasmid-specific primer, 5'-AGCGTCAGACCCCGTAGAAA-3') was paired with a middle-forward mutation primer to synthesize the second half. Mutated amino acids and the sequences of the mutagenic primers are listed in Supplemental Tables I and II. The PCR amplifications were performed in a GeneAmp PCR system 9700 (Applied Biosystems, Tokyo) with 5 ng of pBR-OASA2 plasmid (Tozawa et al., 2001) as the template in a final volume of 50 µL, containing 1x Pyrobest buffer II, 0.2 mM of each deoxynucleoside triphosphate, 0.025 units of Pyrobest DNA polymerase (TaKaRa, Shiga, Japan), and 0.2 µM of sense and anti-sense primers. The PCR conditions were 20 successive cycles of denaturation at 98°C for 15 s, annealing at 60°C for 35 s, and elongation at 72°C for 3 min, followed by a final elongation step at 72°C for 10 min. These half-fragments bearing overlapping sequences introduced by the two middle primers were then mixed together and subjected to three cycles of denaturation at 98°C for 15 s, annealing at 60°C for 30 s, and elongation at 72°C for 3 min. Finally, the mutated DNA fragment produced in the first step was amplified in a further PCR using an outside-forward primer (OASA2-N49) and a nested outside-reverse primer (5'-GGAGAAAGGCGGACAGGTAT-3'). The PCR conditions were 20 cycles of denaturation at 98°C for 15 s, annealing at 60°C for 35 s, and elongation at 72°C for 3 min, followed by a final elongation step at 72°C for 10 min. These fragments were used as the template DNA for split-primer PCR (Sawasaki et al., 2002). OASA2 derivatives bearing double mutations were constructed by the introduction of a second mutation directly to the above-mentioned PCR products, which were single mutants, by using the same PCR-mediated mutation procedure.
Cell-Free Protein Synthesis with the Wheat-Embryo Extracts
Wheat-embryo extracts were kindly provided by T. Shibui at Zoe Gene (Yokohama, Japan). For the first and second screenings, cDNAs encoding OASA2 mutants were constructed by split-primer PCR (Sawasaki et al., 2002) with PCR fragments as templates, and the amplified PCR fragments were directly subjected to in vitro transcription for mRNA preparation. For kinetic analyses of the OASA2 mutants, mutagenized cDNA, encoding S126F, Y367A, A369L, S126F/L530D, Y367A/L530D, or A369L/L530D, was cloned into the pEU3b expression vector (Sawasaki et al., 2002), and was confirmed to have the desired DNA sequences without any additional mutations. In this cloning, mutations were introduced as described above using the upstream forward primer (OASA2N-His, 5'-AAAACTAGTATGcaccatcatcatcatcatTGCTCCGCGGGGAAGCC-3'; the SpeI site is underlined and the His6 sequence is in lowercase print) or the downstream reverse primer (OASA2-C, 5'-AAAGTCGACTGAGAGAGACTCTATTCCTTGTC-3'; the SalI site is underlined) in combination with specific primers for each mutation. In vitro mRNA preparation and cell-free protein synthesis with a wheat-embryo extract were performed as described previously (Madin et al., 2000; Kanno et al., 2004). Synthesized OASA2 proteins were purified as described (Kanno et al., 2004). Enzyme reconstitution for kinetic studies of the AS -subunit, OASA2 derivatives, was performed as described previously with the AS -subunit, the OASB1N58 protein (Kanno et al., 2004).
Enzyme Assay and Kinetic Characterization
The AS activity was measured as described (Kanno et al., 2004). For screening, the NH4+-dependent AS activity of the AS -subunit was assayed at 32°C for 1 h in a 100-µL reaction mixture, containing 20 mM Tris-HCl, pH 8.3, 10 mM MgCl2, 0.5 mM chorismate (Sigma-Aldrich, St. Louis), 100 mM NH4Cl, and 5 µL of test sample. For the kinetic analysis, the Gln-dependent AS activity of the complexes was assayed at 32°C for 30 min with 5 mM Gln as an amino donor, instead of 100 mM NH4Cl, in the reaction mixture. The Km and Vmax values were estimated by measuring the anthranilate production in 100-µL reaction mixtures containing 10 nM of OASA2 wild type or its mutants, as well as 160 nM of OASB1N58 and various concentrations of chorismate (25–1,000 µM), in the presence of 5 mM Gln. The IC50, defined as the concentration, which inhibited enzyme activity by 50% under the assay conditions, was estimated from an examination of the various concentrations of Trp (0, 1, 2.5, 5, 10, 25, 50, 100, 250, and 500 µM) in the presence of 200 µM chorismate.
Expression of OASA2 Derivatives in Yeast
To express the OASA2 derivatives in yeast (Saccharomyces cerevisiae), their ORFs were cloned into the pYES2 vector (Invitrogen, Carlsbad, CA). The OASA2N49 cDNA, corresponding to the mature forms of the OASA2 proteins (Kanno et al., 2004), was generated by PCR with the following primer pairs: sense primer, 5'-AAAGGTACCATGTGCTCCGCGGGGAAGCC-3' (KpnI site underlined) and antisense primer, 5'-AAAGAATTCTGAGAGAGACTCTATTCCTTGTC-3' (EcoRI site underlined). The pBR-OASA2 (Tozawa et al., 2001) and mutated OASA2 PCR products, prepared as described above, were utilized as templates. The resulting PCR products were digested with KpnI and EcoRI and cloned into the corresponding sites of the pYES2 vector. The nucleotide sequences of the constructs were confirmed by DNA sequencing. Saccharomyces cerevisiae strain 16395 (MAT trp2::kanMX his31 leu20 lys20 ura30) was purchased from Open Biosystems (Huntsville, AL). Media preparation, transformation and Gal induction were performed as described previously (Kaiser et al., 1994).
Analysis of Soluble Trp in Yeast Cells
Yeast transformants were isolated as single colony on a synthetic complete medium plate (lacking uracil) supplemented with 2% Glc and then were streaked on synthetic complete medium agar plates (lacking uracil and Trp) supplemented with 2% Gal, and cultivated at 30°C for 2 d. The yeast cells (20 mg) were scraped from the agar plate, collected into a 1.5-mL Eppendorf tube, and suspended in 105 µL of distilled water. The cell suspensions were heated at 100°C for 20 min, homogenized with 595 µL of a chloroform:methanol mixture (5:12, v/v), and centrifuged at 20,000g for 10 min, and then the supernatants were pooled. The supernatants were mixed with 175 µL of distilled water and 263 µL of chloroform, vortexed vigorously for 30 s, and centrifuged at 20,000g for 10 min, and then the aqueous phase was collected. The aqueous solutions were dried by evaporation, and the resultant pellets were dissolved in 200 µL of 10 mM NaOH. The Trp content in each sample was examined with Waters Alliance HPLC FLD System 2695 (Waters, Milford, MA) on an Xterra RP18 column (4.6 x 150 mm). Trp was detected by fluorescence with excitation at 278 nm and emission at 348 nm. The Trp concentration in the sample was estimated from the peak area in the HPLC analysis, by comparison with an authentic Trp dilution series.
Rice Transformation and Trp Analysis
The OASA2 full-length cDNA and its mutant genes were generated by PCR as described above with an outside-forward primer, 5'-AAAACTAGTATGGAGTCCATCGCCGCCGCCACG-3' (the underlined letters are SpeI site), an outside-reverse primer, 5'-AAAGTCGACTGAGAGAGACTCTATTCCTTGTC-3' (SalI site underlined), middle reverse primer (Y367A-R or L530D-R; Supplemental Table II), middle-forward primer (Y367A-F or L530D-F; Supplemental Table II), and a template plasmid vector, pBR-OASA2, containing full-length ORF of the OASA2 (Tozawa et al., 2001). To clone the gene of interest with Sse8387I restriction site present in the binary T-vector, we have modified the pEU3b cell-free expression vector to incorporate the Sse8387I sites into both ends of the multiple cloning site of the construct. This vector, pEU3s, allows transfer of target cDNA into the Sse8387I site of the T-vector, pUB-Hm (Urushibara et al., 2001). The resulting PCR products were digested with SpeI and SalI and cloned into the corresponding sites of the pEU3s vector, resulting in pEU3s-OASA2(wt), pEU3s-OASA2(Y367A), and pEU3s-OASA2(Y367A/L530D). The nucleotide sequences of the constructs were confirmed by DNA sequencing. For rice transformation, the full-length wild-type or mutated OASA2 cDNA was subcloned into pUB-Hm to generate T-vector, which also contains hygromycin phosphotransferase as a selectable marker gene (Urushibara et al., 2001), resulted in pUB-OASA2(wt)-Hm, pUB-OASA2(Y367A)-Hm, pUB-OASA2(Y367A/L530D)-Hm, respectively (Supplemental Fig. 1). Callus induction from seeds of rice (Oryza sativa) L. cv Nipponbare, transformation, and selection of transformed cells were performed as described (Tozawa et al., 2001; Yamada et al., 2004). Isolated transformant calli and nontransformed calli were cultivated on 2N6 medium plate (Yamada et al., 2004) for 2 weeks at 28°C, and total RNA was isolated as described previously (Tozawa et al., 2001). The 5 µg of RNA was subjected to electrophoresis through a 1.2% agarose-formaldehyde gel, and the separated RNA molecules were transferred to a nylon membrane and subjected to hybridization with an OASA2 riboprobe (Tozawa et al., 2001). Probe labeling with digoxigenin-dUTP, hybridization, and immunological detection were performed with DIG Northern Starter kit (Roche Diagnostics, Tokyo). Transgenic and nontransgenic rice calli were cultured on 2N6 media plate for 2 weeks at 28°C (Tozawa et al., 2001). A total of 100 mg of fresh calli were ground in liquid nitrogen, extracted with 0.5 mL of chloroform:methanol:water (5:12:3, v/v/v), and centrifuged at 20,000g for 10 min, and then the supernatants were pooled. The supernatant was mixed with 375 µL of distilled water and 250 µL of chloroform, vortexed vigorously for 30 s, centrifuged at 20,000g for 10 min, and the aqueous phase was collected. Soluble Trp was detected with the same procedure for Trp analysis in yeast cells as described above.
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ACKNOWLEDGMENTS
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We thank Prof. Yaeta Endo for valuable discussions and critical suggestions. We also thank Dr. Tatsuro Shibui and his colleagues for wheat-embryo extracts, and Ms. Chikako Mikami for technical assistance.
Received March 15, 2005;
returned for revision May 6, 2005;
accepted May 9, 2005.
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FOOTNOTES
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1 This work was supported by the Japan Science and Technology Agency for Core Research for Evolutional Science and Technology. 
[w] The online version of this article contains Web-only data.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.062885.
* Corresponding author; e-mail tozaway{at}ccr.ehime-u.ac.jp; fax 81–89–927–8276.
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