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Applied and Environmental Microbiology, November 2003, p . 6399-6404, Vol . 69, No . 11

Use of Bacterial {gamma}-Glutamyltranspeptidase for Enzymatic Synthesis of {gamma}-D-Glutamyl Compounds

Hideyuki Suzuki,* Shunsuke Izuka, Hiromichi Minami, Nobukazu Miyakawa, Sayaka Ishihara, and Hidehiko Kumagai

Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan

Received 9 May 2003/ Accepted 7 August 2003


   ABSTRACT

 
An enzymatic method for synthesizing various {gamma}-D-glutamyl compounds efficiently and stereospecifically involving bacterial {gamma}-glutamyltranspeptidase (EC 2.3.2.2) with D-glutamine as a {gamma}-glutamyl donor was developed . With D-glutamine as a {gamma}-glutamyl donor instead of L-glutamine in {gamma}-glutamyltaurine synthesis, by-products such as {gamma}-glutamylglutamine and {gamma}-glutamyl-{gamma}-glutamyltaurine were not synthesized and the yield of {gamma}-glutamyltaurine dramatically increased from 25 to 71% . It was also shown that the purification could be simplified without these {gamma}-glutamyl by-products . The possibility of synthesizing various {gamma}-D-glutamyl compounds was also shown .


   INTRODUCTION

 
{gamma}-Glutamyltranspeptidase (GGT) (EC 2.3.2.2) catalyzes the hydrolysis of {gamma}-glutamyl compounds such as glutathione and the transfer of their {gamma}-glutamyl moieties to other amino acids and peptides (27) . The reactions catalyzed by GGT proceed via a {gamma}-glutamyl enzyme intermediate involving the hydroxyl group of Thr-391 (9) . If the intermediate is subjected to nucleophilic attack by water, the reaction is hydrolytic, releasing glutamic acid . If the intermediate is subjected to nucleophilic attack by amino acids and peptides, the reaction is a transpeptidation yielding new {gamma}-glutamyl compounds . By employing various {gamma}-glutamyl acceptors, we can synthesize various {gamma}-glutamyl compounds using GGT . The pH optima for these reactions are very different (25) . Therefore, by adjusting the pH of the reaction mixture, we can make GGT catalyze the transpeptidation reaction selectively . {gamma}-Glutamyl compounds are very attractive, because (i) compounds that are not very soluble in water become much more soluble with {gamma}-glutamylization (5); (ii) the {gamma}-glutamyl linkage is resistant to peptidases in serum, and some {gamma}-glutamyl compounds can possibly be used as prodrugs specific for the organs that express GGT (8, 10, 18, 30); and (iii) some {gamma}-glutamyl compounds taste good and thus can be used as food additives (21, 22) . We have already developed and reported efficient methods for synthesizing various {gamma}-L-glutamyl compounds using Escherichia coli GGT (13-17, 21, 22, 26) . The characteristics of our methods are as follows . (i) L-Glutamine, which is less expensive than glutathione, can be used as a {gamma}-glutamyl donor . (ii) No energy source, such as ATP, is required because GGT is a transferase and not a synthetase . (iii) Since E . coli GGT exhibits a broad substrate specificity for {gamma}-glutamyl acceptors, various {gamma}-glutamyl compounds can be synthesized . (iv) Since E . coli GGT can be purified from overproducing strains by means of a simple two-step method (1, 23), a large amount of GGT is readily available .

During the development of a method for the enzymatic production of various {gamma}-glutamyl compounds, we found that the yield of a {gamma}-glutamyl compound with a {gamma}-glutamyl acceptor such as taurine was not very high . This was mainly because nonnegligible amounts of by-products, such as {gamma}-glutamylglutamine and {gamma}-glutamyl-{gamma}-glutamyltaurine, were formed (26) . It has been reported that GGT cannot utilize D-amino acids as {gamma}-glutamyl acceptors (27-29) . Therefore, by using D-glutamine as a {gamma}-glutamyl donor instead of L-glutamine, we expected that neither {gamma}-D-glutamyl-D-glutamine nor {gamma}-D-glutamyl-{gamma}-D-glutamyltaurine would be synthesized and that the yields of the expected compounds would increase . Moreover, there is a latent demand for {gamma}-D-glutamyl compounds . For example, L-theanine ({gamma}-L-glutamylethylamide) is known as the major umami component of Japanese green tea (20) . It is known that there is a positive correlation between a grade of Japanese green tea and its concentration of theanine (4, 19) . Ekborg-Ott et al . (3) reported that D-theanine also exists in tea leaves and that it has a taste similar to that of L-theanine . However, they did not compare the threshold concentrations of L- and D-theanine for perceiving the taste . If D-theanine tastes as strong as L-theanine, D-theanine could be used instead of L-theanine to improve the taste of tea . It has also been reported that {gamma}-D-glutamyl amino acids, such as {gamma}-D-glutamyltaurine, have an antagonistic effect against excitatory amino acids (2, 11) . We speculate that the relaxing effect of L-theanine (12) might possibly be observed for D-theanine with a much lower dosage . Therefore, an effective method for producing {gamma}-D-glutamyl compounds is necessary to obtain a large amount of these {gamma}-D-glutamyl compounds for testing on animals . It was advantageous to develop an enzymatic method involving bacterial GGT for synthesizing {gamma}-D-glutamyl compounds .


   MATERIALS AND METHODS

 
Chemicals and enzyme.
{gamma}-D-Glutamyltaurine and L-theanine were purchased from Tocris Cookson (St . Louis, Mo.) and Tokyo Kasei (Tokyo, Japan), respectively, and used as standards . {gamma}-L-Glutamyl-L-glutamine was from Sigma Chemical (St . Louis, Mo.), and D-glutamine, taurine, and ethylamine were from Nacalai Tesque (Kyoto, Japan) . E . coli K-12 strain SH642 (23), which harbors pUC18 with the E . coli GGT gene, was grown at 20°C (6, 24) in Luria-Bertani broth containing 100 µg of ampicillin/ml . Its periplasmic fraction was prepared (24), and the overproduced GGT was purified to homogeneity by ammonium sulfate fractionation and chromatofocusing as described previously (23) .

Measurement of GGT activity.
GGT activity was measured as described previously (25) by using {gamma}-L-glutamyl-p-nitroanilide and glycylglycine as substrates . One unit of enzyme was defined as the amount of the enzyme that released 1 µmol of p-nitroaniline per min from {gamma}-L-glutamyl-p-nitroanilide through the transpeptidation reaction .

Measurement of amino acids and {gamma}-glutamyl compounds.
Amino acids and {gamma}-glutamyl compounds were measured by high-pressure liquid chromatography (HPLC) as described previously (21, 26) . Briefly, {gamma}-glutamyltaurine and {gamma}-glutamyl-{gamma}-glutamyltaurine were measured with an HPLC instrument (model L-7100; Hitachi, Tokyo, Japan) equipped with a Cosmosil SC18-AR column (10 by 250 mm) (Nacalai Tesque) . Both {gamma}-glutamyltaurine and {gamma}-glutamyl-{gamma}-glutamyltaurine were eluted with water containing 0.05% trifluoroacetic acid at a flow rate of 2.5 ml/min and detected by measuring the absorbance at 213 nm . Amino acids and other {gamma}-glutamyl compounds were measured with an HPLC instrument (model LC-9A; Shimadzu, Kyoto, Japan) equipped with a Shim-pack amino Na column (Shimadzu), with gradient elution at 60°C at a flow rate of 0.6 ml/min . The gradient of the mobile phase was formed with buffer A (66.6 mM citrate, 1% perchloric acid, 7% ethanol, pH 2.8) and buffer B (200 mM citrate, 200 mM boric acid, 0.12 N NaOH, pH 10) . The concentration of buffer B was kept at 0% until 9 min . It was linearly increased to 7% from 9 to 13 min, to 8% from 13 to 17.2 min, and then to 11% . o-Phthalaldehyde was used as the detection reagent, and the fluorescence was detected with a fluorescence detector (model RF-535; Shimadzu) as the absorbance at 450 nm, with excitation at 348 nm .

Large-scale production and purification of {gamma}-D-glutamyltaurine and D-theanine.
{gamma}-D-Glutamyltaurine and D-theanine were synthesized and purified as described previously (21, 26) with slight modification . Briefly, the synthesis of {gamma}-D-glutamyltaurine was carried out with 15 ml of the reaction mixture comprising 200 mM Gln, 200 mM taurine, and 0.2 U of GGT/ml, pH 10, at 37°C for 5 h . The reaction mixture was applied to a column (30 ml) of Dowex 1 x 8, which had been prepared as the CH3COO- form . The column was washed with 200 ml of water and 200 ml of 5 N acetic acid . {gamma}-Glutamyltaurine was eluted with a mixture of formic acid, acetic acid, and water (1:2:16 by volume) . The fractions that contained only {gamma}-glutamyltaurine were saved and lyophilized . The synthesis of D-theanine was carried out with 22-ml of the reaction mixture comprising 150 mM Gln, 150 mM ethylamine, and 0.2 U of GGT/ml, pH 10, at 37°C for 5 h . The reaction mixture was applied to a column (50 ml) of Dowex 50W X 8, which had been prepared as the Ca2+ form . Theanine was eluted with water, and the fractions containing theanine were collected and lyophilized . Theanine was dissolved with water and applied to a column (30 ml) of Dowex 1 X 8, which had been prepared as the Cl- form . Theanine was eluted with water, and the fractions containing only theanine were collected and lyophilized .

NMR and polarimeter analysis.
Purified {gamma}-D-glutamyltaurine and D-theanine were subjected to 1H nuclear magnetic resonance (NMR) and polarimeter analysis as described previously (21, 26) . Purified {gamma}-D-glutamyltaurine (5 mg) and D-theanine (5 mg) were dissolved in 0.5 ml of D2O and analyzed with a Bruker 500-MHz spectrometer, and then the spectrum was compared with that obtained with {gamma}-D-glutamyltaurine and L-theanine purchased from commercial sources . Purified {gamma}-D-glutamyltaurine (5 mg) and D-theanine (10 mg) were dissolved in distilled water (1.2 and 1.0 ml, respectively) and then analyzed with a Jasco DIP-1000 polarimeter (Nippon Bunko, Tokyo, Japan) .


   RESULTS AND DISCUSSION

 
Effect of the use of D-glutamine on the yield of {gamma}-glutamyltaurine.
The optimum reaction conditions for the synthesis of {gamma}-L-glutamyltaurine were 200 mM L-glutamine, 200 mM taurine, and 0.2 U of GGT/ml, pH 10 (26) . Therefore, the yields of enzymatic synthesis of {gamma}-glutamyltaurine using L-glutamine and D-glutamine were compared under these conditions . The reaction mixture (total volume, 1 ml) was incubated at 37°C, an aliquot of it being withdrawn every hour to measure the concentrations of amino acids and {gamma}-glutamyl compounds . When L-glutamine was used as the {gamma}-glutamyl donor (Fig . 1A), it decreased greatly after 1 h of incubation and about 45 mM {gamma}-glutamyltaurine accumulated . In addition to {gamma}-glutamyltaurine, {gamma}-glutamyl-{gamma}-glutamyltaurine and {gamma}-glutamylglutamine were formed . The concentration of {gamma}-glutamyltaurine remained almost constant thereafter . That of {gamma}-glutamylglutamine decreased in 1 h, while that of {gamma}-glutamyl-{gamma}-glutamyltaurine increased gradually until glutamine and {gamma}-glutamylglutamine had been completely consumed . Since a significant amount of the by-product {gamma}-glutamyl-{gamma}-glutamyltaurine was formed after 5 h of incubation and since glutamine was wasted when L-glutamine was used as the {gamma}-glutamyl donor (Fig . 1A), the rate of conversion of L-glutamine to {gamma}-glutamyltaurine was unsatisfactory (Table 1) .


FIG . 1 . Enzymatic synthesis of {gamma}-glutamyltaurine with L-glutamine (A) or D-glutamine (B) as the {gamma}-glutamyl donor . The reaction mixture for the synthesis of {gamma}-L-glutamyltaurine comprised 200 mM L-glutamine, 200 mM taurine, and 0.2 U of GGT/ml at pH 10 and 37°C . That for the synthesis of {gamma}-D-glutamyltaurine comprised 200 mM D-glutamine, 200 mM taurine, and 0.2 U of GGT/ml at pH 10 and 37°C . Samples were withdrawn at the indicated times, and the concentrations of {gamma}-glutamyl compounds and amino acids were determined . Solid circles, {gamma}-glutamyltaurine; open circles, {gamma}-glutamylglutamine; solid triangles, {gamma}-glutamyl-{gamma}-glutamyltaurine; solid squares, glutamine; open squares, taurine.

 

TABLE 1 . Comparison of the yields of {gamma}-glutamyltaurine and theanine after a 5-h reaction with L- or D-glutamine as the {gamma}-glutamyl donora

 
On the other hand, when D-glutamine was used as the {gamma}-glutamyl donor (Fig . 1B), no by-product, such as {gamma}-glutamyl-{gamma}-glutamyltaurine or {gamma}-glutamylglutamine, was formed . As a consequence, the yield of {gamma}-glutamyltaurine increased dramatically (Table 1) .

Effect of the use of D-glutamine on the yield of theanine.
The optimum reaction conditions for the synthesis of L-theanine were 200 mM L-glutamine, 1,500 mM ethylamine, and 0.4 U of GGT/ml, pH 10 (21) . The yields of enzymatic synthesis of theanine with L-glutamine and D-glutamine were compared under these conditions . When L-glutamine was used as the {gamma}-glutamyl donor (Fig . 2A), it decreased to 1/10 of the original level after 1 h of incubation and about 100 mM theanine accumulated, which increased to 120 mM after 2 h of incubation . But its concentration remained unchanged thereafter . In this case, {gamma}-glutamylglutamine was formed as a by-product . When D-glutamine was used as the {gamma}-glutamyl donor (Fig . 2B), no by-product was formed and the yield of theanine increased (Table 1) . However, the yield of D-theanine was only slightly higher than that of L-theanine, possibly because the yield of L-theanine was much higher than that of {gamma}-L-glutamyltaurine . The rate of synthesis of theanine with D-glutamine was slower than that with L-glutamine, and about 50 mM D-glutamine remained, even after 5 h of incubation, and thus the yield may improve with longer incubation .


FIG . 2 . Enzymatic synthesis of theanine with L-glutamine (A) or D-glutamine (B) as the {gamma}-glutamyl donor . The reaction mixture for the synthesis of L-theanine comprised 200 mM L-glutamine, 1,500 mM ethylamine, and 0.4 U of GGT/ml at pH 10 and 37°C . That for the synthesis of D-theanine comprised 200 mM D-glutamine, 1,500 mM ethylamine, and 0.4 U of GGT/ml at pH 10 and 37°C . Samples were withdrawn at the indicated times, and the concentrations of {gamma}-glutamyl compounds and amino acids were determined . Solid circles, theanine; open circles, {gamma}-glutamylglutamine; solid squares, glutamine.

 
Purification of {gamma}-D-glutamyltaurine and D-theanine.
{gamma}-D-Glutamyltaurine and D-theanine were synthesized and purified as described in Materials and Methods . When {gamma}-D-glutamyltaurine was purified, the purification step with reverse-phase HPLC was omitted . This was because {gamma}-D-glutamyltaurine had already been isolated in the purification with a Dowex 1 X 8 column . {gamma}-Glutamyl-{gamma}-glutamyltaurine was not produced when D-glutamine was used as the {gamma}-glutamyl donor, and thus it was not necessary to separate it from {gamma}-glutamyltaurine by reverse-phase HPLC . We emphasize that this is another merit of utilizing D-glutamine as a {gamma}-glutamyl donor .

Purified samples were identified as {gamma}-D-glutamyltaurine and D-theanine by 1H NMR and polarimeter analysis as shown in Fig . 3 and 4 and Table 2 .


FIG . 3 . 1H NMR spectra of {gamma}-D-glutamyltaurine obtained from a commercial source (top) and that synthesized and purified in this study (bottom) . They were measured in D2O with a Bruker 500-MHz spectrometer . Each peak is assigned to a proton of {gamma}-glutamyltaurine and denoted by a letter.

 

FIG . 4 . 1H NMR spectra of L-theanine obtained from a commercial source (top) and D-theanine synthesized and purified in this study (bottom) . They were measured in D2O with a Bruker 500-MHz spectrometer . Each peak is assigned to a proton of theanine and denoted by a letter.

 

TABLE 2 . Results of polarimeter analysis

 
Substrate specificity of E . coli GGT for {gamma}-glutamyl acceptors with D-glutamine as the {gamma}-glutamyl donor.
The transpeptidation reaction was performed with various {gamma}-glutamyl acceptors . The amounts of {gamma}-glutamyl compounds formed versus time were measured by HPLC, and the initial velocities were calculated . The relative activity for {gamma}-glutamyl acceptors is expressed as the relative initial velocity in Table 3 . Under the reaction conditions used, L-tryptophan, taurine, L-methionine, L-phenylalanine, and L-histidine were good acceptors . Therefore, the acceptor specificity with D-glutamine as the {gamma}-glutamyl donor is almost the same as that with {gamma}-L-glutamyl-p-nitroanilide (25) . When ethylamine was used as the acceptor, the production of theanine was not detectable under the reaction conditions used . This may be because the reaction pH, and the concentrations of GGT and ethylamine used for this assay were very different from those used for the production of D-theanine, with which the yield of D-theanine was reasonably good .


TABLE 3 . Substrate specificity for {gamma}-glutamyl acceptors with D-glutamine as the {gamma}-glutamyl donor

 
Comparison of the affinities of GGT for D- and L-{gamma}-glutamyl compounds.
The Km values for {gamma}-L-glutamyl-p-nitroanilide, {gamma}-D-glutamyl-p-nitroanilide, L-glutamine, and D-glutamine were 12.5, 137, 8.3, and 278 µM, respectively, as measured by means of the hydrolysis reaction . Although the Km values for {gamma}-D-glutamyl compounds are much higher than those for {gamma}-L-glutamyl compounds, they are small enough for the enzymatic production of {gamma}-glutamyl compounds by means of our method, for which several hundred millimolar D-glutamine is used .

In conclusion, {gamma}-D-glutamyl compounds can be synthesized efficiently and stereospecifically by employing bacterial GGT and D-glutamine . Furthermore, by using D-glutamine as the {gamma}-glutamyl donor instead of L-glutamine, we can minimize the synthesis of by-products and possibly simplify the purification procedure .

 


   ACKNOWLEDGMENTS

 
We thank Kazuhiro Irie, Graduate School of Agriculture, Kyoto University, for the NMR and polarimeter analysis and for fruitful discussions .

This work was supported by a grant-in-aid for scientific research, no . 13660090, to H.S . from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by research funds from the Society for Research on Umami Taste to H.S .


   FOOTNOTES

 
* Corresponding author . Mailing address: Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan . Phone: 81-75-753-6278 . Fax: 81-75-753-6275 . E-mail: hideyuki{at}lif.kyoto-u.ac.jp .


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