Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Suzuki, H. Articles by Kumagai, H. Search for Related Content PubMed PubMed Citation Articles by Suzuki, H. Articles by Kumagai, H. Agricola Articles by Suzuki, H. Articles by Kumagai, H.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, October 2004, p. 6324-6328, Vol. 70, No. 10
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.10.6324-6328.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

A Single Amino Acid Substitution Converts -Glutamyltranspeptidase to a Class IV Cephalosporin Acylase (Glutaryl-7-Aminocephalosporanic Acid Acylase)

Hideyuki Suzuki,^* Chinatsu Miwa, Sayaka Ishihara, and Hidehiko Kumagai

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

Received 28 January 2004/ Accepted 11 June 2004

ABSTRACT

The aspartyl residue at position 433 of -glutamyltranspeptidase of Escherichia coli K-12 was replaced by an asparaginyl residue. This substitution enabled -glutamyltranspeptidase to deacylate glutaryl-7-aminocephalosporanic acid, producing 7-aminocephalosporanic acid, which is a starting material for the synthesis of semisynthetic cephalosporins.

The effective production of 7-aminocephalosporanic acid (7-ACA) is a matter of concern in the pharmaceutical industry because it is a starting material for the synthesis of semisynthetic cephalosporins, which are the best-sold antibiotics worldwide, with global sales of $8.3 billion of $466.3 billion of the total pharmaceutical market in 2003. Semisynthetic cephalosporins are made by the modification of the side chains of positions 3 and 7 of 7-ACA, which are commercially supplied mainly by the chemical deacylation of cephalosporin C (CPC), produced by the fungus Acremonium chrysogenum. However, the chemical process requires several complicated steps using toxic compounds and produces a lot of chemical wastes. The innovation of an enzymatic process involving two enzymes has recently become the new deacylation process of CPC on an industrial scale (12). This enzymatic process involves no toxic raw materials, proceeds under mild reaction conditions, and reduces waste significantly. In this process, D-amino acid oxidase converts CPC to 7ß-(5-carboxy-5-oxopentanamido)-cephalosporanic acid, followed by autoconversion to glutaryl-7-ACA (GL-7-ACA). Cephalosporin acylase then deacylates GL-7-ACA to 7-ACA. The critical enzyme of this bioprocess is cephalosporin acylase, and extensive screening for this enzymatic activity is extremely important. However, this enzyme has been found only in a limited number of bacterial strains.

There is a surprisingly high similarity between a class IV cephalosporin acylase (GL-7-ACA acylase) (7) and -glutamyltranspeptidase (GGT; EC 2.3.2.2). Seventy-six of about 580 amino acid residues were completely conserved in the GGTs for which the amino acid sequences are known. Of these 76 amino acid residues, 58 were also conserved in class IV cephalosporin acylases. GGT catalyzes the hydrolysis of -glutamyl compounds and the transfer of their -glutamyl residues to other amino acids and peptides (18). Although the -glutamyl group [NH₂CH(COOH)CH₂CH₂CO] and the glutaryl group [CH₂(COOH)CH₂CH₂CO] have similar chemical structures and the linkage between the 7-amino group of 7-ACA and the glutaryl group is an amide linkage (Fig. 1), GL-7-ACA deacylating activity was not detected in Escherichia coli GGT. Asp-433 of E. coli GGT is one of the residues that are completely conserved in GGTs but not in class IV cephalosporin acylases. This residue corresponds to that of human GGT, postulated to interact with the amino group of the -glutamyl residue of its substrate (2), and the residue is Asn in class IV cephalosporin acylases. Therefore, in this study, the GGT enzyme with the D433N mutation (GGT D433N) was obtained and studied.

View larger version (12K): [in this window] [in a new window]

FIG. 1. Reactions catalyzed by cephalosporin (GL-7-ACA) acylase and GGT. Reaction 1 is the reaction catalyzed by cephalosporin (GL-7-ACA) acylase, reaction 2 is the hydrolysis reaction catalyzed by GGT, and reaction 3 is the transpeptidation reaction catalyzed by GGT.

The bacterial strains, plasmids, and oligonucleotide used in this study are listed in Table 1. Single-stranded DNA of pSH1248 was obtained by superinfecting strain SH1277 with M13KO7. The D433N substitution was generated to obtain pCM2 using single-stranded pSH1248 as a template and phosphorylated oligonucleotide GGT D433N as a mutagenic primer by the method of Kunkel et al. (8). The correctness of the DNA sequence of pCM2 was confirmed by the method of Sanger et al. (11) as modified by Katayama et al. (6). The 0.9-kb HindIII fragment of pCM2 was ligated with the 5.4-kb HindIII fragment of pSH253 to obtain pCM3. The 3.2-kb SmaI-SphI fragment was then ligated with the 4.0-kb EcoRV-SphI fragment of pBR322 to obtain pCM7. Strain SH641 was transformed with pCM7, and strain CM9 was obtained.

View this table: [in this window] [in a new window]

TABLE 1. Bacterial strains, plasmids, and oligonucleotide used in this study

The mutant GGT protein (GGT D433N enzyme) was isolated from the periplasmic fraction (17) of strain CM9 as described previously (15). The GGT activities of the wild-type GGT and mutant GGT D433N enzyme were measured as described previously using -glutamyl-p-nitroanilide and glycylglycine as substrates (16). Their protein concentrations were measured by the method of Lowry et al. (10). No transpeptidation activity was detected for GGT D433N enzyme, but its hydrolysis activity was 0.32 U/mg, about 82% of the wild-type enzyme. This might be because the -glutamyl residue of the -glutamyl enzyme intermediate formed at Thr-391 (3) wobbles without the hydrogen bond with Asp-433, and it is difficult for a -glutamyl acceptor to make the nucleophilic substitution from a distinct direction. On the other hand, water could attack its carbonyl carbon because of its small size.

GL-7-ACA, used to measure the activity for deacylating GL-7-ACA, was synthesized essentially by the method of Shibuya et al. (13) from glutaric anhydride and 7-ACA, purchased from Wako Pure Chemical Industry (Osaka, Japan) and Aldrich Chemical, respectively. The synthesized sample was identified as GL-7-ACA by nuclear magnetic resonance (NMR) and fast atom bombardment mass spectrometric analyses. Succinyl-7-ACA, adipyl-7-ACA, and adipyl-7-aminodeacetoxycephalosporanic acid (7-ADCA) were synthesized similarly from the corresponding acid anhydrides and 7-ACA or 7-ADCA, except for adipic anhydride, which was incubated at 60°C to dissolve in acetone.

The deacylating activity of GL-7-ACA was measured as follows. The standard reaction mixture was 50 mM Tris-HCl (pH 8.73), 2 mM GL-7-ACA, and 0.1 mg of enzyme per ml. The reaction was initiated by the addition of the enzyme solution, and the reaction mixture was incubated at 37°C. Part of the reaction mixture (200 µl) was subtracted and mixed with the same volume of 3.5 N CH₃COOH to terminate the reaction. The concentrations of both GL-7-ACA and 7-ACA after filtration through a membrane filter (pore size, 0.2 µm) were measured by a high-performance liquid chromatograph (model LC-10; Shimadzu, Kyoto, Japan) equipped with an Inertsil ODS-3 column (5 mm by 250 mm; GL Sciences, Tokyo, Japan) with gradient elution at a flow rate of 1 ml/min and at 40°C. The gradient of the mobile phase was formed with buffer A (0.05% trifluoroacetic acid) and buffer B (acetonitrile containing 0.05% trifluoroacetic acid). The concentration of buffer B was linearly increased to 50% from 0 to 25 min, kept at 50% until 34 min, and then decreased to 0% from 34 to 35 min. Both GL-7-ACA and 7-ACA were detected with a UV detector (model SPD-10AVP; Shimadzu) at an absorbance of 280 nm.

Enzymatic deacylation of GL-7-ACA with the GGT D433N enzyme.

Whether the side chain at position 7 of GL-7-ACA could be deacylated with the GGT D433N enzyme was examined. The reaction mixture containing the GGT D433N enzyme was incubated at 37°C for 3 h and analyzed by high-performance liquid chromatography (HPLC). As shown in Fig. 2, a new peak with a retention time corresponding to the 7-ACA purchased from a commercial source was observed after a 3-h incubation. This suggested that the deacylation of GL-7-ACA was catalyzed by the GGT D433N enzyme.

View larger version (21K): [in this window] [in a new window]

FIG. 2. Chromatograms of HPLC analysis of the reaction mixture of deacylation of GL-7-ACA by the GGT D433N enzyme. The original reaction mixture (2 mM GL-7-ACA, 50 mM Tris-HCl [pH 8.73]) was incubated with 0.1 mg of the GGT D433N enzyme per ml at 37°C for 0 h (A) and 3 h (B). The numbers shown on the left side of the chromatograms are the retention times in minutes. GL-7-ACA eluted at 18.4 min, and 7-ACA purchased from a commercial source eluted at 10.0 min. The structures of GL-7-ACA (a) and 7-ACA (b) are shown to the right of the chromatogram.

Optimum pH of the deacylating reaction.

Since the reaction of GGT is very much influenced by the pH of the reaction mixture, its effect was determined (Fig. 3). The optimum pH of the deacylating reaction of GL-7-ACA as a substrate was 8.73, which is identical to the optimum pH of the transpeptidation reaction using -glutamyl-p-nitroanilide and glycylglycine as substrates (16), and not to that of the hydrolysis reaction using -glutamyl-p-nitroanilide as a substrate (3).

View larger version (17K): [in this window] [in a new window]

FIG. 3. Effect of reaction pH on the deacylation reaction of GL-7-ACA. The optimum pH for the GGT D433N enzyme was determined in the following buffers: 50 mM potassium phosphate buffer (filled circles), 50 mM Tris-HCl (open circles), and 50 mM NH4Cl-NH4OH buffer (filled squares).

Identification of the reaction product as 7-ACA.

Although the retention time of the reaction product by HPLC was the same as that of 7-ACA, it was not evident that the peak indicated as the reaction product was in fact 7-ACA. The reaction was performed for 3 h with 730 ml of the reaction mixture. The reaction mixture was applied to a column (30 ml) of Dowex 1X8, which was prepared as the CH₃COO^– form. The column was washed with 150 ml of water. The possible 7-ACA was then eluted with 0.5 N CH₃COOH and lyophilized. The sample was dissolved in water and subjected to reverse-phase HPLC with a Cosmosil 5C18-AR-II column (20 mm by 250 mm) (Nacalai Tesque, Kyoto, Japan). The fraction containing possible 7-ACA was lyophilized, and 18.3 mg of possible 7-ACA was obtained. The possible 7-ACA was subjected to NMR analysis (Bruker 500-MHz spectrometer). As shown in Fig. 4, the NMR chart of the sample exactly matched that of 7-ACA purchased from a commercial source. This indicates that the GGT D433N enzyme indeed catalyzed the deacylating reaction of GL-7-ACA to produce 7-ACA.

View larger version (16K): [in this window] [in a new window]

FIG. 4. 1H-NMR spectrum of 7-ACA obtained from a commercial source (A) and that obtained as described in the text (B). Both were measured in D2O (2 mg/0.7 ml) with a Bruker 500-MHz spectrometer. Each peak is assigned to a proton of 7-ACA and assigned a letter.

Substrate specificity of the GGT D433N enzyme.

The substrate specificity of the GGT D433N enzyme was measured using synthesized substrates and cephalosporin C purchased from a commercial source (Sigma-Aldrich). The GGT D433N enzyme cleaved GL-7-ACA at the rate of 0.12 µmol/min/mg, but did not cleave CPC, succinyl-7-ACA, adipyl-7-ACA, or adipyl-7-ADCA at a detectable rate. In this study, the detection limit was 6 x 10^–5 µmol/min/mg. The cephalosporin (GL-7-ACA) acylases previously reported had some activity to CPC derivatives other than GL-7-ACA (7). Therefore, the substrate specificity of the GGT D433N enzyme is very strict in comparison with those of other enzymes. This may be because the original structure of GGT was not suited to cleave CPC derivatives.

Kinetic parameters of the GGT D433N enzyme.

The kinetic parameters of the GGT D433N enzyme using GL-7-ACA as a substrate were determined. The K_m value for GL-7-ACA was 0.198 mM, and the k_cat value was 0.122 s^–1. The K_m and k_cat values of the class IV cephalosporin (GL-7-ACA) acylases previously reported were 6.1 mM and 17 s^–1, respectively (4), and those of GL-7-ACA acylase from Pseudomonas sp. strain GK16 were 1.05 mM and 9.48 s^–1, respectively (9). The k_cat value of the GGT D433N enzyme was low, but the K_m was very low. Therefore, the k_cat/K_m value was not very different from those of other enzymes.

In conclusion, the single amino acid substitution of GGT, Asp-433 to Asn, converted GGT to cephalosporin (GL-7-ACA) acylase. Although the k_cat value has to be increased by introducing other mutations before it can be applied in the pharmaceutical industry, this study showed that the ggt gene could be a new source for cephalosporin (GL-7-ACA) acylase. Since GGT is known to be distributed widely in biological organisms and eukaryotic GGTs are 500- to 1,000-fold more active than E. coli GGT, it is anticipated that we could find a much better ggt gene from other organisms.

ACKNOWLEDGMENTS

We thank Kazuhiro Irie, Graduate School of Agriculture, Kyoto University, for the NMR and mass spectrometric analyses and helpful discussions.

This work was supported in part by a grant-in-aid for scientific research (grant 15580061) to H.S. from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. C.M. and S.I. are supported by the 21st Century COE Program of the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

 
* Corresponding author. Mailing address: Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Oiwake-cho, 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.

FOOTNOTES

Present address: Research Institute of Agricultural Resources, Ishikawa Agricultural College, Ishikawa-gun, Ishikawa 921-8836, Japan.

REFERENCES

1
1. Claudio, J. O., H. Suzuki, H. Kumagai, and T. Tochikura. 1991. Excretion and rapid purification of -glutamyltranspeptidase from Escherichia coli K-12. J. Ferment. Bioeng. 72:125-127.[CrossRef]
2
2. Ikeda, Y., J. Fujii, N. Taniguchi, and A. Meister. 1995. Human -glutamyl transpeptidase mutants involving conserved aspartate residues and the unique cysteine residue of the light subunit. J. Biol. Chem. 270:12471-12475.[Abstract/Free Full Text]
3
3. Inoue, M., J. Hiratake, H. Suzuki, H. Kumagai, and K. Sakata. 2000. Identification of catalytic nucleophile of Escherichia coli -glutamyltranspeptidase by -monofluorophosphono derivative of glutamic acid: N-terminal Thr-391 in small subunit is the nucleophile. Biochemistry 39:7764-7771.[CrossRef][Medline]
4
4. Ishiye, M., and M. Niwa. 1992. Nucleotide sequence and expression of the cephalosporin acylase gene of a Pseudomonas strain. Biochim. Biophys. Acta 1132:233-239.[Medline]
5
5. Joyce, C. M., and N. D. Grindley. 1984. Method for determining whether a gene of Escherichia coli is essential: application to the polA gene. J. Bacteriol. 158:636-643.[Abstract/Free Full Text]
6
6. Katayama, T., H. Suzuki, T. Koyanagi, and H. Kumagai. 2000. Cloning and random mutagenesis of the Erwinia herbicola tyrR gene for high-level expression of tyrosine phenol-lyase. Appl. Environ. Microbiol. 66:4764-4771.[Abstract/Free Full Text]
7
7. Kim, Y., K.-H. Yoon, Y. Khang, S. Turley, and W. G. J. Hol. 2000. The 2.0 A crystal structure of cephalosporin acylase. Structure 8:1059-1068.[Medline]
8
8. Kunkel, T. A. 1985. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc. Natl. Acad. Sci. USA 82:488-492.[Abstract/Free Full Text]
9
9. Lee, Y. S., H. W. Kim, K. B. Lee, and S. S. Park. 2000. Involvement of arginine and tryptophan residues in catalytic activity of glutaryl 7-aminocephalosporanic acid acylase from Pseudomonas sp. strain GK 16. Biochim. Biophys. Acta 1523:123-127.[Medline]
10
10. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275.[Free Full Text]
11
11. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5467.[Abstract/Free Full Text]
12
12. Schmid, A., F. Hollmann, J. B. Park, and B. Buhler. 2002. The use of enzymes in the chemical industry in Europe. Curr. Opin. Biotechnol. 13:359-366.[CrossRef][Medline]
13
13. Shibuya, Y., K. Matsumoto, and T. Fujii. 1981. Isolation and properties of 7ß-(4-carboxybutanamido)cephalosporanic acid acylase-producing bacteria. Agric. Biol. Chem. 45:1561-1567.
14
14. Suzuki, H., and H. Kumagai. 2002. Autocatalytic processing of -glutamyltranspeptidase. J. Biol. Chem. 277:43536-43543.[Abstract/Free Full Text]
15
15. Suzuki, H., H. Kumagai, T. Echigo, and T. Tochikura. 1988. Molecular cloning of Escherichia coli K-12 ggt and rapid isolation of -glutamyltranspeptidase. Biochem. Biophys. Res. Commun. 150:33-38.[CrossRef][Medline]
16
16. Suzuki, H., H. Kumagai, and T. Tochikura. 1986. -Glutamyltranspeptidase from Escherichia coli K-12: purification and properties. J. Bacteriol. 168:1325-1331.[Abstract/Free Full Text]
17
17. Suzuki, H., H. Kumagai, and T. Tochikura. 1986. -Glutamyltranspeptidase from Escherichia coli K-12: formation and localization. J. Bacteriol. 168:1332-1335.[Abstract/Free Full Text]
18
18. Taniguchi, N., and Y. Ikeda. 1998. -Glutamyl transpeptidase: catalytic mechanism and gene expression. Adv. Enzymol. Rel. Areas Mol. Biol. 72:239-278.[Medline]

---

Applied and Environmental Microbiology, October 2004, p. 6324-6328, Vol. 70, No. 10
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.10.6324-6328.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Yamada, C., Kijima, K., Ishihara, S., Miwa, C., Wada, K., Okada, T., Fukuyama, K., Kumagai, H., Suzuki, H. (2008). Improvement of the Glutaryl-7-Aminocephalosporanic Acid Acylase Activity of a Bacterial {gamma}-Glutamyltranspeptidase. Appl. Environ. Microbiol. 74: 3400-3409 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Suzuki, H. Articles by Kumagai, H. Search for Related Content PubMed PubMed Citation Articles by Suzuki, H. Articles by Kumagai, H. Agricola Articles by Suzuki, H. Articles by Kumagai, H.