Applied and Environmental Microbiology, October 2004, p . 6324-6328, Vol . 70, No . 10

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 .

 
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 .

 
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 .

 
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) .

 
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 .

 
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 .

FOOTNOTES

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

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