Biotechnol. Prw. 1993, 9, 323-331
323
Molecular Cloning of the y-Glutamyltranspeptidase Gene from a Pseudomonas Strain Masayuki Ishiye, Mitsuo Yamashita, and Mineo Niwa’ Product Development Laboratories, Fujisawa Pharmaceutical Co., Ltd., 1-6, a-Chome, Kashima, Yodogawa-ku, Osaka 532, Japan
y-Glutamyltranspeptidase (GGT) was purified from a Pseudomonas sp. strain A14. The purified enzyme was found to be composed of two nonidentical subunits with molecular weights of 39 000 and 22 000 and had a p1 of >8.6. The partial N-terminal amino acid sequences of both subunits and some proteolytic fragments were determined. Using mixed oligonucleotides designed from the partial amino acid sequences as hybridization probes, one cosmid clone which contained the GGT gene was isolated from a Pseudomonas sp. strain A14 cosmid genome library, and the DNA sequence of the GGT gene was determined. The nucleotide sequence and the protein sequence analysis revealed that GGT was synthesized as a precursor protein of 575 amino acids and then processed to mature enzyme, presumably after removal of a signal peptide. Comparison of the predicted amino acid sequence of Pseudomonas GGT with published results for Escherichia coli K-12 and rat kidney GGTs shows that the protein sequence of Pseudomonas GGT is 51% and 33 % identical to the E . coli and rat GGT sequences, respectively. Higher similarity is observed among the small subunits, which have been thought to have a binding site for the y-glutamyl residue. Expression of the cloned Pseudomonas GGT gene in E . coli was subjected to Western blot analysis using antibody raised against the purified GGT. This suggested that processing of the precursor protein to its subunits is temperature-dependent, because the amount of mature GGT protein was increased when the culture was performed a t low temperature.
E. coli K-12 GGT has been deduced from ita DNA sequence
Introduction y-Glutamyltranspeptidase(GGT, EC 2.3.2.2) is a widely distributed enzyme that catalyzes the hydrolysis of glutathione and a variety of y-glutamyl compounds and the transfer of their y-glutamyl residue to amino acids or peptides (Meister et al., 1981; Tate and Meister, 1981). Many mammalian GGTs have been purified and characterized in detail (Tate and Meister, 1985). GGT is composed of two nonidentical subunits which derived from a single chain precursor protein (Tate and Meister, 1985; Barouki et al., 1984). The cDNAs of rat, human, and porcine GGTs have been cloned and sequenced (Sakamuro et al., 1988; Goodspeed et al., 1989; Rajpert-De Meyts et al., 1988;Papandrikopoulou et al., 1989). Although their deduced amino acid sequences are highly conserved, irrespective of their origins, it is suggested that there are some differences between these enzymes with respect to their catalytic and immunological properties (Tate et al., 1988). Recently, a new gene coding for the GGT-related protein was cloned from a human placental cDNA library using a GGT probe. Although the deduced amino acid sequenceof the GGT-related protein exhibited only about 40% similarity with human GGT, it showed ‘GGT-like” activity (Heisterkamp et al., 1991). These results indicate that structurally related, but not identical, enzymes are able to hydrolyze the unusual y-glutamyl bond. On the other hand, bacterial GGTs have also been purified from Proteus mirabilis, Bacillus sp., and Escherichia coli and characterized (Nakayama et al., 1984a;Hwang and Oishi, 1985; Suzuki et al., 1986a). The amino acid sequence of
* Author to whom correspondence should be addressed. Telephone: 06-390-1148.Fax: 06-304-1192. 8756-7938/93/3009-0323$04.00/0
(Suzuki et al., 1989). Cephalosporin acylases, which hydrolyze the acyl side chain of cephalosporin C or 7-@-(4-~arboxybutanamido)cephalosporanicacid to yield 7-aminocephalosporanicacid, are very important bacterial enzymes for the production of cephalosporin antibiotics (Matauda et al., 1987a). We have noticed that a type of cephalosporinacylaseand GGT might be related gene products, judging from the recently published predicted amino acid sequences and subunit compositions (Matsuda et al., 1987131, so that it is reasonable to speculate that both enzymes exhibit similar substrate specificity. To elucidate the catalytic properties of the bacterial GGTs, and to compare them with cephalosporin acylases,we searched novel bacterial GGTs which have catalytic properties different from those of previously characterized bacterial GGTs. We have isolated from soil a strain of Pseudomonirs sp., designated as A14, from which a new GGT has been purified. As our interesta lie in dissecting the relationship between the structure and function of GGT, especially its substrate specificity, we have cloned and sequenced the Pseudomonas GGT gene in preparation for more detailed analysis, such as site-directed mutagenesis. In the present article, we describe the purification, characterization, and determination of partial amino acid sequences of Pseudomonas sp. strain A14 GGT, and we also describe the molecular cloning and sequencing of the corresponding gene and the expression of the cloned gene in E. coli. In addition, we compare the deduced amino acid sequence of Pseudomonas GGT with those of other GGTs.
0 1993 American Chemical Society and American Institute of Chemical Engineers
Biotechnol. Pro&
324
Materials and Methods Materials. Pseudomonas sp. strain A14 was used as the source of enzyme and DNA. This strain was isolated from soil and selected as a specific GGT producer. E. coli strain HBlOl (Boyer and Roulland-Dussoix, 1969) was used for cloning and expression studies, and JM109 (Yanisch-Perron et al., 1985) was used as a host for M13 phage containing recombinant DNA. Restriction endonucleases and other enzymes used for DNA manipulations were purchased from Toyobo (Osaka, Japan) or Takara Shuzo (Kyoto, Japan); plasmid pHC79 was from Boehringer Mannheim GmbH, and pHSG298 was from Takara Shuzo. Plasmid pTTQ8 and radiolabeled nucleotides were obtained from Amersham. Acromobacter protease I (API) was purchased from Wako Pure Chemicals (Osaka,Japan). Nitrocellulose filter was purchased from Schleichelr & Schuell. Horseradish peroxidase conjugated goat antirabbit antibody and 4-chloro-1-naphthol were obtained from Seikagaku Kogyo (Tokyo, Japan) and Bio-Rad, respectively. CM-Sephadex C50, Phenyl-Sepharose CL4B, and Sephacryl S-200 were obtained from Pharmacia LKB Biotechnology Inc. CM-Toyopearl650M and ButylToyopearl were obtained from Toso (Tokyo, Japan). A Cosmosil5C4-300 reversed-phase high-performance liquid chromatography (HPLC) column (4.6 X 50 mm) and L-yglutamic acid p-nitroanilide (GlupNA) were purchased from Nacalai Tasque (Kyoto, Japan). 6-Diazo-boxo-~norleucine (DON) and L-azaserine were the products of Sigma. All other chemicals used were reagent grade. Purification of the GGT from Pseudomonassp.A14. Pseudomonas sp. strain A14 was grown in 10L of Sauton's medium (Bovarnick, 1942)containing 1% L-glutamic acid at 30 "C for 16 h with aeration. Cells were harvested by centrifugation (10 OOOg for 30 min) and stored at -80 "C until use. Frozen cell paste (210 g) was suspended in 2 vol (v/w) of 0.1 M potassium phosphate buffer (pH 7.01, and the cells were disrupted by sonication at 4 "C. The supernatant was obtained by centrifugation at 15 OOOg for 15min. This supernatant was treated with poly(ethy1ene imine) (0.35 % final concentration) and centrifuged at 15000g for 30 min. The resulting supernatant was concentrated by ammonium sulfate precipitation (70 ?6 saturation). The precipitate was dissolved in 0.05 M sodium acetate buffer (pH 6.0, buffer A) and dialyzed against the same buffer. The dialyzed solution was adsorbed to CM-Sephadex C50 (1.5 L) previously equilibrated with buffer A. The mixture was gently stirred for 2 h at 4 "C, and the resin was removed using a glass filter. After it was washed with equilibrating buffer, the resin was suspended in an equal volume of buffer A containing 1 M NaCl and stirred for 1 h. The eluate was collected by filtration and concentrated by ammonium sulfate precipitation (70% saturation). The precipitate was collected by centrifugation, dissolved in 0.05 M potassium phosphate buffer (pH 7.0, buffer B), made 30% saturated with ammonium sulfate, and then dialyzed against buffer B containing 30% saturated ammonium sulfate. The dialyzed protein solution was applied to a PhenylSepharose CL-4B column (2.5 X 30 cm) equilibrated with buffer B containing 30 % saturated ammonium sulfate; after it was washed with the same buffer, the enzyme was eluted with buffer B. Active fractions were pooled and concentrated by ammonium sulfate precipitation. The precipitate was dissolved in buffer A, dialyzed against the same buffer, and then applied to a CM-Toyopearl column (2.5 X 15 cm) equilibrated with same buffer. The enzyme was eluted using a linear gradient of 0-0.6 M NaCl in buffer A. The pooled active fractions were filtered, by
1993, Vol. 9, No. 3
serial application of 5-mL aliquots, through a Sephacryl S-200 column (1.5 X 100 cm) equilibrated with buffer B. The enzyme was eluted with the same buffer at a flow rate of 12 mL/h. Ammonium sulfate was added to the pooled active fractions up to 30% saturation. This mixture was applied to a column of Butyl-Toyopearl (1.5 X 15 cm) equilibrated with buffer B containing ammonium sulfate (30 5% saturation) and was eluted with a linear gradient of ammonium sulfate (304%) in buffer B. Finally, the active fractions were applied to a column of Sephacryl S-200 as described above. All operations were performed at 4 "C. Amino Acid Analysis. The purified Pseudomonas GGT was applied to a preparative SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis)(15% ) gel to separate the two subunits. The protein bands were located by brief staining with Coomassie Brilliant Blue, and each of the subunits was recovered by electroelution. A part of each subunit was then digested with Achromobacter protease I (API) (Masaki et al., 1981) in 0.1 M Tris-HC1 (pH 8.0) in the presence of 0.01% SDS (enzyme: peptide ratio of 1:200) at 37 "C for 5 h. The resulting peptides were resolved by a C-4 reversed-phase column (Cosmosil 5C4-300, 4.6 X 50 mm) using a 40-min linear gradient of acetonitrile from 15 to 60% containing 0.1% trifluoroacetic acid (TFA) at a flow rate of 1 mL/min. Several peaks were collected and concentrated by a Speed Vac concentrator. The N-terminal amino acid sequences of collected peptide fragments were determined by using an Applied Biosystems Model 470A gas-phase protein sequencer. Construction of the Genomic Library. Chromosomal DNA from Pseudomonas sp. strain A14 was prepared as described (Mondello, 1989). Total DNA of Pseudomonas sp. A14 (5 pg) was partially digestedwith Sau3AI to achieve an average fragment size of 3G40 kilobasepairs (kb).These fragments were ligated into the BamHI site of the cosmid vector pHC79 (Hohn and Collins, 1980) (1pg) using T4 DNA ligase, and the ligation mixture was packaged in vitro using a commercially available extract (Stratagene's packaging extract Gigapack Gold) according to the recommendation of the manufacturer and transfected to E. coli HBlOl which had been grown in the presence of 0.2% maltose. Screening and Isolation of the Pseudomonas GGT Gene. Oligonucleotideprobes (see below) were end labeled with [y-32PlATP(3000 mCi/mmol) using T4 polynucleotide kinase and were used to screen the library. Colonies containing recombinant cosmids were transferred to nitrocellulose filters as described (Maniatis et al., 1982). The filters were prehybridized at 42 "C for 2 h in 6X SSC, 5X Denhardt's reagent (Maniatis et al., 1982),0.1% SDS, and 50 pg/mL denatured salmon sperm DNA. Hybridization was performed for 18 h at 42 "C with the same solution plus the radiolabeled probe (2 X lo6 cpm/mL). After hybridization, the filters were washed twice at room temperature in 6X SSC-O.l% SDS for 30 min and then at 42 "C for 5 min. The filters were air dried and exposed to X-ray film (Kodak X-Omat) at -70 OC for 70 h with an intensifying screen. Several positive clones were identified through colony hybridization. One of these plasmids, named p3148, was used for further characterizations. Southern blot analysis revealed that p3148 contained a 9.2-kb BglII fragment, which hybridized to all oligonucleotide probes used. To reduce the size of p3148 (ca. 42 kb), p3148 was digested with BglII and religated. Plasmid p29 (15 kb) was one of the plasmids thus obtained and consisted of a part of the
325
Bbtechrwl. Rug., 1993, Vol. 9, No. 3
Table I. Summary of Pseudomonas sp. A14 GGT Purification
steps
extract CM-Sephadex PhenylSepharoseCL-4B CM-to yopearl Sephacryl S-200 Butyl-Toyopearl 2nd Sephacryl S-200
specific activity purifiprotein activity (units/mg yield cation (mg) (units) ofprotein) (%) (-fold) 0.026 100 1.0 10450 272 1800 171 0.095 62 3.6 0.31 45 12 392 122 45.6 6.3 3.6 2.9
120 54 44 37
2.70 8.58 12.3 13.0
B
A
44 20 16 14
103 330 473 500
1 2 pl
.
+-7.35
k ., ,,--6.55 --
31.O
"
S
'
2
3
97.4 66.2 45.0
-;
4-
.
7 -5.85 '.L
vector (pHC79) and 9.2 kb of the Pseudomonas DNA insert, which contained the entire Pseudomonas GGT gene. Southern Blot Analysis. Chromosomal DNA or cosmid DNAs were digested with various restriction endonucleases, and the digested DNA fragments were separated by agarose gel electrophoresis and then transferred to nitrocellulose filter. Hybridization was performed as described (Maniatis et al., 1982). Construction of the Expression Vectors. The 3.2kb SphI fragment, which contains the coding region of the Pseudomonas GGT gene, was isolated from p29 and cloned into the SphI site of pHSG298 (Takeshita et al., 1987) to yield pGGT298A14. A 2.6-kb BalIIHindIII fragment was isolated from pGGT298A14and ligated with an expression vector pTTQ8 (Stark, 1987), which was previously digested with SmaI and HindIII, to yield Pseudomonas GGT gene expression vector pMI470. In this vector, the coding region of the GGT gene, excluding the 5'-terminal34 base pairs (bp), was located downstream from the tac promoter and the ribosome-binding site of pTTQ8. Therefore, the N-terminal12 amino acids of the GGT (MKNQTFSKALLA)were replaced by 4 amino acids (MNSP) (Figure 6). Expression of the Cloned GGT Gene in E. coli. E. coli HBlOl harboring pMI470 and cultured ovemight at 30 "C was diluted by 50-fold in 5 mL of fresh L-broth containing 50 pg/mL ampicillin and was then cultured a t 37 or 30 "c. When the optical density at 600 nm (&m) of the culture medium reached 0.6-0.8, isopropyl 8-Dthiogalactopyranoside (IPTG) was added to a final concentration of 1mM, and the culture was continued at the same temperature for 16 h. Total cellular proteins were separated by SDS-PAGE (15%), and expression of the Pseudomonas GGT was verified by Western blot analysis. Antibody Production and Western Blot Analysis. Purified GGT (100 pg in Complete Freund's adjuvant) was injected intradermally in New Zealand White rabbits, and a booster injection (100 pg of GGT in Incomplete Freund's adjuvant) was administered every 2 weeks. The animals were bled .8 weeks later, and the antiserum was stored at -20 "C. SDS-PAGE was performed using the system described by Laemmli (1970). Protein bands were transferred from the gel to nitrocellulose filter by the procedure of Towbin et ai. (1979) and were detected using horseradish peroxidase conjugated goat anti-rabbit antibody and 4-chloro-1-naphthol. Enzyme Assay. GGT activity was determined spectrophotometrically, as described (Nakayama et al., 1984b), using L-y-glutamic acid p-nitroanilide as the y-glutamyl donor. The reaction mixture (final volume, 0.4 mL), consisting of 2.5 mM L-y-glutamic acid p-nitroanilide, 50 mM Tris-HC1 (pH 8.9), 60 mM acceptor molecule, and enzyme, was incubated at 37 "Cfor 10 min. The reaction was stopped by the addition of 0.8 mL of 3.5 N acetic acid,
3-8.65 (J-8.15
M W M 1
(W
'
3-5.20 .. * -4.55 , -3.50
21.5 14.4
Figure 1. SDS-PAGE and isoelectric focusing of purified GGT and its subunits. (A) About 1 pg of purified GGT was subjected to a PhastGel IEF 3-9, using PhastSystem (Pharmacia). Lane 1 contained GGT, and lane 2 contained p l marker protein. (B) Purified GGT (2 pg, lane l),isolated large subunit (2 pg, lane 2), and isolated small subunit (2 pg, lane 3) were analyzed by SDSPAGE (15% gel). Lane M contained molecular weight marker proteins. Protein bands were stained with Coomassie Brilliant Blue. Table 11. Peptide Sequences Derived from the Pseudomonas GGTb sequence large subunit 1. AXQAPVGAENGXXVXA 2. AGISQEIXPGVPXXEGS 3. GASTTGYLAVGVPGTVXXME 4. TRQQLISPAITLADKGFVLEQGDVDM 5. GFVLEQGDVDMLXT small subunit 6. 'M'HYSIVDKDGNXVSVXYXLNDXF 7. VGVPNMYGLIQGEANAIGPGRRPL
location0 25-40 358-374 121-141 151-176 166-179 376-399 423-446
The locations indicated are derived from the predicted protein sequence. Fragments 1 and 6 are the intact large and small subunits, respectively. Other fragments are derived from API-digested subunits. X denotes an identified amino acid. Underlined sequences in fragments 3,4,6, and 7 were used to design hybridization probes (Figure 2).
and the amount of liberated p-nitroaniline was determined from the optical density a t 410 nm. One unit of the enzyme activity is defined as the amount of the enzyme required to release 1pmol of p-nitroaniline per minute from L-Yglutamic acidp-nitroanilide at 37 "C. The specificactivity is defined as units/mg of protein. The protein content was determined by the method of Bradford (1976) using bovine serum albumin as the standard. Other Methods. All oligonucleotides were chemically synthesized using an Applied BiosystemsDNA synthesizer (Model 380A) according to the manufacturer's recommended protocols and were purified on a C-18 reversedphase column. Restriction fragments were subcloned into bacteriophage vectors, M13mp18and M13mp19, and DNA sequence was determined by the method of Sanger et al. (1977) using an Applied Biosystems Model 370A automated DNA sequencer according to the manufacturer's recommended protocols. The p l value of purified GGT was measured on a Pharmacia isoelectric focusing gel (PhastGel IEF 7-9),using a Phast System (Pharmacia LKB Biotechnology Inc.) following the manufacturer's instructions. Amino acid sequences were compared and aligned by the method of Lipman and Pearson (1985) using the PRINAS software (Mitsui Knowledge Industry Co.).
Biotechnol. hog., 1993, Vol. 9, No. 3
326 LS 2 6 m e r ( 6 4 m i x t u r e s )
Protein sequence
GlyTyrLeuAlaValGlyValProGly
Posible codons
5 ’ GGCTACCTCGCCGTCGGCGTCCCCGG G T G G G G G G
3’
Degenarate Probes
3 ’ CCGATGGAGCGGCAGCCGCAGGGGCC A C C C c c
5’
LS 3 2 m e r ( 6 4 m i x t u r e s ) Protein sequence
AspLysGlyPheValLeuGluGlnGlyAspVal
Posible codons
5 ’ GACAAGGGCTTCGTCCTCGAACAAGGCGACGT T G G G G G G T
3’
Degenerate Probes
3 ’ CTGTTCCCGAAGCAGGAGCTTGTTCCGCAACA A c c c c G
5’
SS 2 l m e r ( 1 6 m i x t u r e s ) Protein sequence
IleValAspLysAspGlyAsn
Posible codons
5 ’ ATCGTCGACAAGGACGGCAAC G T T G
3’
Degenerate Probes
3 ’ TAGCAGCTGTTCCTGCCGTTG C A A C
5’
SS 4 5 m e r ( 1 2 8 m i x t u r e s ) Protein sequence
ValGlyValProAsnMetTyrGlyLeuIleGlnGlyGluAlaAsn
Posible codons
5 ’ GTCGGCGTCCCCAACATGTACGGCCTCATCCAAGGCGAAGCCAAC 3 ’ G G G G G G G G G G
Degenerate Probes
3 ‘ CAGCCGCAGGGGTTGTACATGCCGGAGTAGGTTCCGCTTCGGTTG 5 ’ C c c C C c c
Figure 2. Design of four degenerate oligonucleotide probes for Pseudomonas GGT based on partial amino acid sequence analysis. The wobble positions of the codons were chosen for G or C.
Results Purification and Characterization of the Pseudomonas GGT . We searched bacterial GGTs which had some properties, such as acceptor specificity, that were different from those previouslydescribed for bacterial GGTs. Since bacterial GGTs hydrolyze GlupNA well in the presence of glycylglycine but have reduced activity in the absence of glycylglycine(Nakayama et al., 1984a;Suzuki et al., 1986a), GGT activity was measured in the presence or absence of glycylglycine. We found out that a Pseudomonassp. strain A14, isolated from soil, produced a novel GGT showing the same activity toward GlupNA with or without glycylglycine. Pseudomonas sp. strain A14 was cultured in Sauton’s medium supplemented with L-glutamic acid (Hwang and Oishi, 1985). The produced GGT was easily purified from total cell lysate as described in Materials and Methods. The results of the purification are summarized in Table I. From 210 g of the cells, about 2.9 mg of pure enzyme was obtained. Pseudomonas sp. strain A14 GGT was determined by Sephacryl 5-200 gel filtration to have a molecular weight of ca. 60 000 and a PIof >8.6 (Figure 1A). The purified enzyme was composed of two nonidentical subunits, one large and one small. Their molecular weights were calculated by SDS-PAGE to be 39 OOO and 22000, respectively (Figure 1B). These results indicate that the native enzyme is composed of one large subunit and one small subunit. Pseudomonas GGT recognized various amino acids as y-glutamyl acceptors. Glycylglycine was a poor acceptor
500 bp
w Signal Large Subunit Small Subunit
Figure 3. Restriction map and sequencing strategies for the Pseudomonas GGT gene. Restriction map of the 3.2-kb SphI fragment is shown at the top of the figure. Arrows below the map indicate the direction and extent of the sequence determined. The predicted physical organization of the protein is also indicated. The enzymes used are BaZI (B),NaeI (N),Sal1 (Sa), Sac1 (Sc), SmaI (Sm), SphI (Sp), and XhoI (XI.
for transpeptidation, whereas L-Met and L-Asn were good acceptors. The optimal pH of hydrolysis and transpeptidation (in the presence of 60 mM L-Met) of GlupNA was about 9-10 for both reactions. The K, value for GlupNA in the hydrolysis was 68 pM. The enzyme activity was completely inhibited by DON and L-azaserine,which are specific GGT inhibitors, a t concentrations of 1 mM. Determination of Amino Acid Sequences. Both the large and small subunits were separated by SDS-PAGE (15% gel) and were electrically eluted from the corresponding gel slices. About 3 nmol of each subunit was digested with API (Masaki et al., 1981) at 37 O C for 5 h, and the digested fragments were separated using a C-4 reversed-phase column. N-Terminal amino acid sequences
Biotechnd. Rw., 1993, Vol. 9, NO. 3
327
GCCCCCAl C G T T T C C G C C C C C A l ~ A ~ ~ A C T C G C A C l C A ~ C ' l ' O h ~ A T C G A A C T C A A C G
CTTACCGCCCAACGCTTTCACGCACTCCGCACATTTGTCCAGTGCCACGCCAATGCGCGT
Ati'I'titiCCCGl'CtiCCCCCACTTTTGACCCATCTTTCCCC,\ATTACTC~ATTTGCGTTACCG
850
860
870
880
890
!IO0
TCCCCCCGCCTCCTGATCTCCGAGATCA~GAATATTCTCGAAGCCTATCCGATGAAA~AA SerGlyGlyValVallleCysG~ulleMelA~nlleLeuCluG~yTyrProMeLLysGlu 910
920
930
940
9sn
9eo
CTCGCCTATCACTCGCCCCAGGGCGTGCACTACACCATCGAAGCCATGCGTCACGCCTAC
T C T T G T T T C C C C A C G G A A T T T T T C C C C A C C A C G C C A A ~ A G ~ 4 C A T C G C C C C LeuGlyTyrHlsSerAIaGInGl~~alHisTyr~hrlhrlleGluAlahetAr~HisAla~yr CCCGACCCTCCCACCCTCAGGCACCCGTCAGTTCtiAGCGAGCTGCCCGTCCGTGCGAAGC G T C C C C C C C C C A A T C C A C C A G T T T C C T C A A A A G A C G IO
20
30
40
50
60
ATCAAAAATCAAACCTTCTCCAAAGCGT~ACTGGCCACAGCCCTGAGCTOTGCG~TGTTC MeLLysAsnClnThrPheSerLySAlaLeuLeuAlaThrAlaLeuSerCysAlaLeuPhe
970
1150
100
110
1010
1020
LeuAspLysAspTyrAlaAlaLysIleArgAlaAla~leAsnProGlnLYsA~aGlY~~e
130 140 150 I60 170 180 CACCACATCGCATCGAAGCTCGGCGTCGAAGTGCTCAAGTCCGGCGGCAACGCGATCGAT
90
1000
1030 1040 1050 1060 1070 IO60 CTCCACAAGCACTACOCCCCOAAOATTCGTGCCGCGATCAAC~CGCAGAAGGCCGGTATC
A090
80
990
~~lA~pArgAsnSerTyrLeuGlyAspProAspPheVa~l,ysAsnPraLeuAla~~sLeu
120
70
980
GTCGACCGCAACAGCTATCTGGGCGACCCGGACTTCGTGAAGAACCCG~TC~CGCATCTG
1100
I110
1120
I I30
1140
TCGCAAGAGATCAAGCCGGCTGTGCCGCCGCATGAAGGCAGCAACACGACGCACTACTCG A,~CGTTCACCCGCCGTCCCAAGCGCCGGTGGtiCGCCGAGAACGGCATGGTCGTGACCGCG A n n ~ a l H ~ s A l a A l a S e r C 1 n A l a P r o V a l G l y A l a G l u A s n G l y M e l V a ~ V a l T h r A l a SerClnGluIleLysProGlyValProProHisCluClySerAsnThrThrHisTyrSer 1160
I170
1180
1190
1200
ATCCTCGACAACCATGCCAATGCCGTGTCCGTCACCTACACGCTCAACGACTGGTTCGGC GlnllislleAlaSerLysValG~yValGluVnlLeuLysSerGlyG~yAsnAlaI~eAsp IleValAspLysAspGlyAsnAlaValSerValTh~TyrThrLeuAsnAapTrp~heGly 1 go 200 210 220 230 240 GCCCCACTGCCCGTGCCCTATGCGCTGGCCGTGGTGTATCCGGCAGCCGGCAACATCGGC
1210
1220
1230
1240
1250
1260
CCGAACCTCATGCCCAACCGCACGGGCGTGCTGCTCAACGACGAGATGGACGACTTCACC
A l a A l a V a l A l a ~ a l C l y T y r A l n L e u A l a V a l ~ a ~ T y r P r o A l a A l a G l y A s n I ~ e G l yAlaLysValMeLAlaAsnOlyThrGlyValLeuLeuAsnAspGluMetAspAspPheThr 250
260
270
280
290
300
GCCCCCGCCTTCATGACGATTCAACTCCCCaACCCCCGCAAGACCTTCCTCGACTTCCGC ClyGlyClyPheMetThrIleGlnLeuAlaAspGlyArgLys~hrPheLeuAspPhaAr~ 310 320 330 340 350 380 CAGAACCCCCCGCTCCCTCCCACCGC~AA~ATGTACCTCGATAAGGACGGCAACGTCATC
GluLysAlaProLeuAlaAlaThrAlaAsnnetTyrLeuAspLysAspGlyAsnValIle 370
3R0
390
400
410
420
AAGCCCCCCTCCACCACCGCCTATCTCGCCCGTGGGTGTGCCGGGCACCGTATCGGGCATG
LysClyAlaScrThrThrGl)'Tyrl.euAlaValGlyValProG1yThrVn1SerCly~et 430
440
450
4 ti0
470
480
4 9n
500
510
520
530
540
1210
1280
1290
I300
1310
1320
TCCAACCTCCGTGTCCCCAACATGTACGGCCTGATTCAGGGCGAAGCCAACGCCATCGGC SerLysValClyValProAsnMetTyrGlyLeuIleGlnGlyGluAlaAsnAla~leGly 1330
1340
1350
1360
1310
1380
CCCGGCCCTCGCCCCCTCTCGTCGATGAGCCCGACCATCGTCACCAAGGATGGCAAGACG ProGlyArgArgProLeuSerSerMetSerProThrlleValThrLysAspGlyLysThr 1390 1400 1410 1420 1430 1440 CTGATGGTCCTCGGTACGCCGGGCGGCAGCCGCATCATTACGGCCACGCTGCTCACGATG
ValMetValValClyThrProGlyGlySerArgll~IleThrAlaThrLeuLeuThrMeL 1450
1460
1470
1480
1490
1500
CACTACGCACCTCACAAGTACGGCACGAAGA~GCGTCAGCAACTGATCTCGCCGGCCATC CTCAACATGATCCACTACGGCATGAACCTGCAAGAAGCCGTGGATGCCCCGCGCTTCCAC C l u T y r A l a A r ~ C l u L y s T p r G ~ y ~ h r L y s T h r A r g G l n G l n L e u ~ l e S e r P r o A ~ ~ ILleeu A s n M e t I l e A s p T y r C l y M e l A s n L e u G l n G l u A l a V a l A s p A l a P t o A r g ~ h e H i s 1510
1520
1530
1540
1550
1560
ThrLeuAlaAspLysClyPheValLeuGluGlnGlyAspValAspMeLLeulrpThrSer
CAGCACTGGATGCCGGAATCGACCAACATCGAAGCGTTTGCCCTGAGCCCGGATACGCAG GlnClnTrpMet~roCluSerThrAsnIleCluAlaPheAlaLeuSer~roAspThrGln
550 560 570 580 590 600 ACCAAGGACTTCGACAAACACCGCGCCAACTCGGGCGCCATCTTCATGAACAAGGGTCAG
AAGATTCTCGACAGCTGCCCCCAGAAGTTTGCCGGCCCGCAGCCCGCGAATCACATCGCT
ACCCTCCCCGACAACCCCTTTGTGCTCGAGCACGGCGACGTGGACATGCTGTGGACGTCG
ThrLYsAsPPheCluLysAspArgAlaAsnSerGlyAlallePheMeLAsnLysG~yGln GI0
620
630
640
650
660
I570
1580
1590
1600
1610
1620
LyslleLeuGluSerTrpClyGlnLyaPheAlaG~yProGlnProA~aAsnH~s~~eAla 1630
1640
1690
1700
1650
I660
1710
1720
1670
I680
CCCTTCCAGCCCCCCCACCCTCTGGTG~AGAAGGATCTCGCGCGCACGCTGCGCCTGATG ProPheClnProClyCluArgLeuValGlnLysAspLeuAlaArgThrLeuArgLeuIl~
GCGATTCTGCTCGCTGCGCCGTCGCTGGGCGGCAAGCCGATCGGCAAGAACCGCTTCTAC AlalleLeu~a.GlyAlaProSerLeuGlyG~YLYsProl~eG~yLYsAsnArgPneTyr
670 680 690 700 710 I20 ACCCCCAACCGCACCCACGGCTTCTACAAGCCTGAAtiT~GCCGACAAGCTCGTGCCCTCG
GGTCCCAACCATCCCCCCCGCAATACGGGTCTCGCGCTCGGGTATTGATCGCTTGATAAC
1730
S c r h l $ L y s C l y T h r A s p C I ~ P h e T y r L ~ s G l y G l u Y a l A ~ a A s p L y s L e u V n l A ~ ~ S e rC l y A l a A s n A s p P r o A r g A r g A s n T h r G l Y L e u A l a L e u G l y T y r * * * 7 30 740 7 50 760 770 780 :\TG~~GCCGCGCGGCGGC~\TCAT~ACGCAGGCTGACCTCGACCACTACAAGACGCGCGAA
~cLL?s~~laGlrClyGiyllelleThrGl~iAlnAspLeuAspGlnTyrL~~ThrAr~Glu 79n eon n I ii 820 830 840 CTCCCCCCCCTCC~~~TCCGAT1'~~CCGCGGC'rATCACGTAGTClCGCCCCCGCCCCCGA~C LeuAlaPro~alGluCysAsPT~rAr~Clvl'~rHisYalValSerAlaPr~ProPr~S~r
Figure 4. Nucleotide sequence and the deduced amino acid sequence of the Pseudomonas GGT gene. Numbering of the DNA sequence starts at the 5'-end of the putative initiation codon. A possible promoter site is denoted by underlining, and the possible ribosome-binding site is boxed. The C-terminal inverted repeat sequences are indicated by facing arrows.
of each subunit and some of their proteolytic fragments were determined (Table 11). Design of Probes. The partial amino acid sequences (Table 11) allowed the design of oligonucleotidesthat were specific for the GGT gene. The underlined sequences of fragments 3, 4, 6, and 7 in Table I1 were used to design four set of degenerate oligonucleotide probes (Figure 2). These probes were designed with a G-C bias in the third position of the codons, as has been found for other Pseudomonas genes (Zylstra and Gibson, 1989). Isolation and Characterization of the GGT Gene. Approximately 2000 colonies from the Pseudomonas strain A14 DNA cosmid library were screened by colony hybridization using 5'-32P-labeledoligonucleotidesas probes. Eleven positive clones were obtained, and three of these clones were further characterized by Southern blot analysis of their plasmid DNAs. Two of these plasmid DNAs (p3148 and p6105) had a common 9.2-kb BglII fragment, which was hybridized with all four sets of probes. To reduce the size of the plasmid, plasmid DNA (~3148,ca. 42 kb) was digested with BgZII and was religated to yield
a plasmid, p29 (15 kb), which consisted of a part of the " i d vector and the 9.2-kb BglII fragment of Pseudomonas DNA. Subsequent Southern blot analysis indicated that the GGTgene was located on a 3.2-kb SphI fragment, and this region of p29 was sequenced. The restriction enzyme map and the sequencing strategy are shown in Figure 3. SequenceAnalysis of the GGT Gene. Figure 4 shows the nucleotide sequence and the deduced amino acid sequence of the Pseudomonas sp. strain A14 GGT gene with its 5'- and 3'-flanking regions. An open reading frame composed of 575 amino acids was identified in the sequenced region by comparison of the amino acid sequence deduced from the nucleotide sequence to the N-terminal amino acid sequences of the various peptides derived from the purified Pseudomonas GGT. The N-termini of the large and small subunits were found at amino acids 25 and 376, respectively. The assignment of the initiation ATG codon is based upon the fact that the following 23 amino acids have some characteristics of a signal peptide (Perlman and Halvorson, 1983). Thus, it
328
Biotechnol. Pmg., 1993,Vol. Q, No. 3 r . I ~ a r g eS u b u n i t Y I K P - T F l R R V i I I . ~ 1 1 S C S C F S S I A . ~ P ~ \ P P V S Y G V E E D V F H P V R ~ K Q G M V I S V D ~ T ~ T Q V G V D l L K E G G73N ~ V
E. coli
**
t
Pseudomonas
* **
*
** *
* *** * * ****
* * * ***
YKNQTFS-KIlLIT\LSCilFN~Hi~SQ~-------------PVGiENGMVVT~QHl.~SKVGVEVLKSGGN~l
* *
Rat kidney
* *
* **
59
M K N R F t V t C l V ~ ~ V L V F V I l C L C l W l P T T S G K P D H V Y S R ~ ~ V ~ T D ~ K R C S E I G R D M t Q E G G S 64 VV 4
D S ~ V ~ V G Y . ~ t ~ V T H P Q ~ C N l G G G G F M L l R S K N G N T T - I I D F R E M ~ P ~ K ~ T R D M F ~ D D Q C N P D S K - K S L T S H L . ~ S G T P G T V ~ G F 1S6t 0. ~ t D K
************ * *** ****** *
**** ** ** * ** * *
* *
* * * ** * **** *
* *
D . i . ~ V ~ V C Y $ t $ V V Y P ~ I C N I ~ G G G F M T l Q t . I D G R K T - F 1 D F R E K S P l ~ i T ~ N M Y l D K D G N V - l K G . ~ S T T G Y ~ ~ V G V P G T V S G M E Y1 ~4 R6 E K
*** *
**** * * *
* * ** *
**
* * ******
*
* * *
D S ~ I i S t t C M G t I ~ ~ H S M G I G G G L F F T l Y U S T T R K ~ E V I N ~ R E M . I P R ~ i N T S M F - N ~ - - - - - S K D - S E E G G l S V I V P G E I R G Y E t S H Q R1 4 6
*
Y G T M P L N K V V Q P ~ F K l I R D G F l V N D ~ l ~ D D 1 K T Y G S E - V L P N H E N S K S l F ~ K E G E P L K K G D T ~ V Q ~ N t . ~ Ki SE Nt GEPMDlE F Y K G T I A
***
** * * **
* *
* * ***
* * *
* *** ** * *
* * **** *
248
Y G T K T R Q Q t I S P $ I T t $ D K G F V L E Q G D V D M t ~ T S T K D - F E K D R S ~ S G $ l ~ M N K G Q P F Q P G E R ~ V Q K D t . ~ R T t R L I S ~ K G T D G F Y K G234 EV~
*
* * * * * * *
* ** *
*
*
*
*
** * *
***
*t
*
E C R t P V $ R t F Q P S I Q 1 ~ R H G F P V G K G - t S R . I t D K K R D I I E K T P ~ L C E - V F C R Q G K V ~ Q E G E T V T M P K L I D T L Q l t ~ Q E G ~ R ~ F Y ~ G233 StT
E Q I I Q E M Q K N G G t l T K E D l ~ ~ Y K ~ V E - R T - P I S G D Y R G Y Q V ~ S M P - P P S S G G f H l V ~ Q I L N l L E ~ F ~ ~ ~ - D M K K Y G F G S S329 D$MQl~~E~
*
* * ** * * * * *
*
***** * * * ******
* ****
** *
**
I*
D K L V ~ S M K ~ G G G I I T Q ~ D t D Q Y K T R E - t . I - P V E C D Y R G Y H V I ' S ~ P - P P S S G G V - V l C E l M N l L E G Y - - - - P ~ K E t G Y H S ~ Q G V H Y T l E . I 315
*
*** * * * *
*
*
* * ** * * * *** * *
*
*
*
**
I Q I V K D I Q E $ G G I M T V E D L N N Y R l - E V l E H P M S I G L - G D S T t Y V P S ~ P L S G P V ~ L l ~ L l L N l L K G Y N F S P K S V ~ T P E Q K $ l T Y H R I V 318 E.~ 1.
r
Small S u b u n i t E K Y I Y ~ D R S E Y l G D P D F V K V P ~ Q i t T N K ~ - - ~ \ K S I I D Q I D I N K S K P S S E I R P G K L . ~ P Y E S N Q T T H Y S V V D K D G N , i V I V T Y T t N T T 4F1G6T
** ** ********* *
* *
** *
*
** * * * **
* *
***** ********
****ti
**
~ R H ~ Y V D R N S Y L G D P D F V K N P L ~ H t l D K D - - Y ~ I K l R S ~ l ~ P Q K ~ G l S Q E I K P G - V P P H E G S N T T H Y S I V D K D G N ~ V S V T Y T4 t0 1N D ~ F G ~
* ** *
**** * *
**
**
* * * * * * **** * * *
t
$1;
F R F S Y I K R - T M L G D P K F V D V S Q V I R N M S S E F Y ~ T Q t R ~ R l T D E T T H P T ~ Y Y E . ~ E F Y L P D D G G - T ~ H L S V V S E D G S ~ V ~ . ~ T S T I N L4 Y0 F5 G S 4
G I V I G E S G I ~ ~ N N Q ~ D D F S S K P G V P N V Y G ~ V G G D ~ ~ ~ V G P N K R P t S S ~ S P T ~ l V V K D G K T ~ t ~ T G S P G G S R l l T - T V t5Q0 ~3 V V ~ S l D Y G L
*
* *** **** * **** *** * * * * * * * * * * * * * * * ** ***** * * ******** * * * * * * * *
K V M I N G T G V L ~ N D E ~ D D F T S K V G V P N M Y G ~ l Q G E \ ~ ~ l G P G R R P L S S M S P T - l V T K D G K T V M V ~ G T P G G S R l I T i T L L T ~ - 1 488 UHIDYG~ I*
* *
* * ******* *
* * * * * * * * * * ** * * * * **** * * * *
*
*
*
Y ~ L S R ~ S G I ~ F N D E M D D F S S - P Y F T N Q ~ G ~ I T S P ~ ~ F I K L G K ~ P L S S M C P S I I V D K D G K V R ~ V V G ~ S G G T Q I T T S ~ ~492 LI-IIUS~UFGY
I r I E I T V ~ P R F H H Q h L P D E L R ~ E ~ G ~ - - S 2 3 " L K L L F ~ ~ G Q K I \ t ~ E \ ~ G S T Q S l ~ V G - ? DE -L VG G-I-S-3 P R S \ D D L T I C Y
* ** ***** * * *
* * **** *
*I
***
**** *
i
*** ***
* **
380
~ L Q E I I D I P R F H Q Q k Y P E S T Y I EI F ~ 1 S P D T Y K I L E S k C Q K F ~ C P Q P i N H I \ I I L ~ G i P S 1 C C K P I C K U R F Y G ~ N D P R R N T G L I L5G7~5
** * * * * * * *
31KR\IEEDRLHNQtLPYTTT\!K
*
*
'*
**
- U ) Q ~ l ' I ~ L ~ " R - I Y h ' l ~ ~ T P - DI IF\ Ql I I V R ' S C C - - - - -
* * *
ti
w ~ I ~ S D S R K G C E - P I C I 368
%
Figure 5. Comparison of the predicted amino acid sequences of the GGT proteins from Pseudomonas sp. A14, E. coli K-12 (Suzuki et al., 1989), and rat kidney (Sakamuro et al., 1988). Identical amino acids are indicated by the ?# symbol. The beginnings of the large and small subunits are indicated. Arrows below the rat GGT sequence indicate the positions interrupted by introns (Rajagopalan et al., 1990). The comparison was done according to the method of Lipman and Pearson (1985). is indicated that the large and small subunits of the mature GGT are derived from a common 575 amino acid precursor protein, from which a putative signal peptide has been removed. A sequence of inverted repeats followed by a T-rich region was found downstream of the gene (Figure 4). This structure resembles p-independent terminators of enteric bacteria (von Hippel et al., 1984). The codon usage of the GGT gene was highly biased. In 84.5 % of the total codons, in this case the number of codons of Met and Trp was excluded, the third variable position were ended in either G or C, which is typical of Pseudomonas genes. The deduced amino acid sequence of the Pseudomonas GGT was compared with the published sequences of E. coli (Suzuki et al., 1989)and rat kidney (corrected sequence by Sakamuro et al. (1988)) GGT (Figure 5). The amino acid sequence identity between Pseudomonas and E. coli GGT is 51% ,and identity between Pseudomonas and rat
renal GGT is 33%. The similarity among the small subunits is higher than that of the large subunits. Expression of Pseudomonas in E. coli. As the entire GGT gene was located on a 3.2-kb SphI fragment, this fragment was cloned into the SphI site of pHSG298, generating plasmid pGGT298A14. The production of Pseudomonas GGT in E. coli using this plasmid failed, and a protein band was not detected by Western blot analysis using antibody raised against GGT (data not shown). Therefore, we constructed a new plasmid, pMI470, in which the coding region of the GGT gene was placed under the control of the tac promoter, as described in Figure 6. E. coli HBlOl harboring pMI470 was grown in L-broth at 37 or 30 "C, and the expression of GGT was induced by the addition of IPTG (1mM) to the culture medium. Total cellular proteins were separated by SDS-PAGE
Bbtdmd.
m.,1993, Vd. 9, NO. 3
329
A
RBS SmaI Rind111 Sph I Plac pCCT298-
RindIII BalSphl I
Bal I Hind111 + Ball (partial) Ball Bindl I I
i
SaaI
t
AindII I
+
RBS (SmaI/BalI)
indI I I
B SmaI/Bal I
Ptac-
R B S
-A T G A A T T C C C C C A C A C C C C T C A C C N
N
S
P
T
A
L
S
Figure 6. Construction of plasmid pMI470. (A) Scheme for the construction of expression vector pMI470. (B)The nucleotide sequence in the initiation site for the expression of the GGT gene. N-Terminal four amino acids were newly created by SmaI-BalI fusion. Ptac, Plac, and rrnBTITe represent tac promoter, lac promoter, and rrnB transcription terminators, respectively. Km*and Ampr represent kanamycin- and ampicillin-resistant genes, respectively. LacIq is the lac repressor gene, and RBS is the ribosomebinding site. (15%), and the produced GGT was localized by immunoblotting (Figure 7). The GGT protein of E. coli HBlOl was not detected by anti-(PseudomonasGGT) under these conditions. When induced at 37 "C with 1mM of IPTG, a band corresponding to molecular weight of ca. 60 0oO was only detected by immunoblotting (Figure 7, lane 1). This protein might be a precursor protein of Pseudomonas GGT. When IPTG induction was performed at 30"C,the relative intensity of this band was reduced, whereas immunoreactive bands corresponding to the large and small subunits of Pseudomonas GGT were increased (Figure 7, lane 3). These results indicate that the large and small subunits were derived from a common precursor protein and that this processing step is temperaturedependent.
1
2
3
4
5
P @
k@
LS
ss
Discussion We have demonstrated in this article that the novel GGT purified from Pseudomonas sp. strain A14 is composed of two distinct subunits (molecular weights of 39 OOO and 22 OOO); that Pseudomonas GGT has acceptor specificity and p l different from other bacterial GGTs, Le., its hydrolysis activitytoward GlupNA does not increase in the presence of glycylglycine, which is a good acceptor for other GGTs (Tate and Meister, 1985;Nakayama et al., 1984a; Suzuki et al., 1986a);and that Pseudomonas GGT is a very basic protein (pl>8.6), whereas other bacterial GGTs are acidic proteins (Nakayama et al., 1984a; Suzuki et al., 1986a).
Figure 7. Western blot analysis of the total cell proteins. About
100pg of total cell proteins were analyzed by SDS-PAGE (15 95 gel). Proteins were transferred to nitrocellulose filter by electroblotting (Towbin et al., 1979) and visualized as described in Materials and Methods. Lane 1: Total cell proteins from the cells transformed by pMI470, after 16 h of induction with IPTG (cultured at 37 "C). Lane 2: Total cell proteins from control cells cultured a t 37 "C. Lane 3 Total cell proteins from the cells transformed by pMI470, after 16 h of induction with IPTG (culture a t 30 "C). Lane 4 Total cell proteins from control cells cultured a t 30 "C. Lane 5 contained purified GGT (2 pg). The putative precursor protein (P), large subunit (LS), and small subunit (SS) are indicated.
Biotechnol. Prog., 1993, Vol. 9, No. 3
330
Pseudomonas GGT gene was isolated from a cosmid library and characterized. The identity of the gene was confirmed by several lines of evidence. First, N-terminal amino acid sequences of several peptide fragments obtained from API-digested GGT subunits were found to be included in the deduced amino acid sequence (Table 11). Second, the deduced amino acid sequence was found to have significant similarity to other GGTs (Figure 5). Third, calculated molecular weights of the large and small subunits (37 353 and 21 400) were found to correspond well to the estimated molecular weights of each of the subunits of GGT (39 000 and 22 OOO), which were determined by SDS-PAGE. Finally, the expression of the Pseudomonas GGT gene in E. coli was confirmed by Western blot analysis, and the large and small subunits and ita putative precursor protein were detected by antibody raised against purified Pseudomonas GGT (Figure 7). Although the E. coli consensus promoter sequence (McClure, 1985) is located upstream from the putative initiation codon (Figure 4), the expression of the Pseudomonas GGT gene in E. coli was very poor (Figure 7). This result indicates that the promoter of the Pseudomonas GGT gene might be inefficiently recognized by E. coli RNA polymerase. The Pseudomonas GGT gene might be translated inefficiently in E. coli,because the potential ribosome-binding sites are not found in the vicinity of the putative initiation codon ATG, but are located far upstream of the coding region (Figure 4). This long distance between the ribosome-binding site and the initiation codon may render the initiation rate of translation low. To elevatethe expressionlevel of the Pseudomonas GGT gene, an expression vector pMI470 was constructed as described in Figure 6 and was introduced into E. coli HB101. The expression of the Pseudomonas GGT gene in E. coli was improved by optimally locating the coding region of Pseudomonas GGT gene downstream from the tac promoter and ribosome-binding site of plasmid pTTQ8 (Figure 7). The amino acid sequences in mammalian GGT are highly conserved throughout the molecule (Papandrikopoulou, 1989). As is in the case of bacterial GGTs, a high degree of similarity was observed between the protein sequences of Pseudomonas and E. coli GGTs, especially between the small subunits. The identity of amino acid sequences between the small bacterial subunits is 58%, whereas that of the large subunits is 48%. A similar tendency is seen between mammalian and bacterial enzymes (Figure 5). This conservation of amino acid sequence between the small subunits is concordant with the results of Tate and Meister (1977),who demonstrated that the binding site of the y-glutamyl residue was located on the small subunit. In the large subunit, there are some regions of low amino acid sequence similarity among the GGTs (Figure 5). According to the results obtained by Rajagopalan et al. (19901, the DNA sequences coding for the corresponding region of rat GGT are interrupted by introns. Thus, it is probable that the large subunit may be divided into functional parts by these regions, because an exon codes for a functional domain. This local sequence diversity in the large subunit may be attributed to the acceptor specificity of the GGT, as described by Tate et al. (1988). They also showed that the acceptor binding site might be formed by both subunits (Tate et al., 1988; Tate and Ross, 1977). It is suggested that these regions may be constitutive elements of the acceptor binding site of the enzyme.
The processingof the precursor protein of Pseudomonas GGT in E . coli was temperature-dependent (Figure 7), like that of penicillin acylase (Oh et al., 1987),which also consists of two nonidentical subunits and is derived from a common precursor protein. In the case of E. coli GGT, production was temperature-dependent, with maximal production being observed at 20-23 "C (Suzuki et al., 1986b; Kumagi et al., 1988). The processing of the precursor of E. coli GGT was also temperature-dependent. When its gene was expressed under the control of the trp promoter, the amount of active GGT was increased at lower temperature (M. Ishiye and M. Niwa, unpublished observations). The amino acid sequences around the processing sites of penicillin acylase (Oh et al., 1987) and the processing sites of GGTs are quite different; thus, it is suggested that there are different processing pathways in E. coli or that the processing of the precursor protein into subunits is an autocatalytic event. Recently, we cloned and purified a novel cephalosporin acylase and showed that this enzyme resembles GGT with respect to amino acid sequence. Interestingly, it exhibited not only cephalosporin acylase activity but also GGT activity (M. Ishiye and M. Niwa, manuscript in preparation). Thus, the nucleotide sequence of the novel GGT gene makes it possible to perform a more detailed analysis of the relationship between the structure and function of the GGT protein and to compare the GGT protein to cephalosporin acylase, especially with regard to their substrate specificity.
Literature Cited Barouki, R.; Finidori, J.; Chobert, M.-N.; Aggerbeck, M.; Laperche, y.; Hanoune, J. Biosynthesis and Processing of y-GlutamylTranspeptidasein HepatomaTissueCulture Cella. J. Biol. Chem. 1984,259,7970-7974.
Bovarnick, M. The Formation of Extracellular &(-)-Glutamic Acid Polypeptide by Bacillus subtilis. J . Biol. Chem. 1942, 145,415-424.
Boyer, H. W.; Roulland-Dueeoix,D. A. ComplementationAnalysis of the Restriction and Modification of DNA in Escherichia coli. J. Mol. Biol. 1969, 41, 459-472. Bradford, M. M. A Rapid and Sensitive Method for the Quantitationof Microgram Quantitiesof Protein Utilizing the Principle of Protein-Dye Binding. Anal. Biochem. 1976, 72, 248-254.
Goodspeed, D. C.; Dunn, T. J.; Miller, C. D.; Pitot, H. C. Human y-GlutamylTranspeptidasecDNA Comparisonof Hepatoma and Kidney mRNA in the Human and Rat. Gene 1989, 76, 1-9.
Heisterkamp, N.; Rajpert-De Meyta, E.; Uribe, L.; Forman, H. J.; Groffen,J. Identification of a y-Glutamylcleaving enzyme related to, but distinct from, y-Glutamyl Transpeptidase. R o c . Natl. Acad. Sci. U.S.A. 1991,88, 6303-6307.
Hohn, B.; Collins, J. A Small Cosmid for Efficient Cloning of Large DNA Fragment. Gene 1980,11,291-298. Hwang, S. Y.; Oishi, K. Purification and Properties of an Extracellular y-Glutamyl Arylamidase from Bacillus sp. strain No. 12. Agric. Biol. Chem. 1986, 49, 3255-3264.
Kumagai, H.; Echigo, T.; Suzuki, H.; Tochikura, T. Synthesis of y-Glutamyl-DOPAfrom L-Glutamine and L-DOPAby yGlutamyltranspeptidase of Escherichia coli K-12. Agric. Biol. Chem. 1988,52,1741-1745. Laemmli, U. K. Cleavage of Structural Proteins During the Assembly of the Head of Bacteriophage T4. Nature 1970, 227,680-685.
Lipman, D. J.; Pearson, W. R. Rapid and Sensitive Protein Similarity Searches. Science 1986,227, 1435-1441. Maniatis, T.; Fritah, E. F.; Sambrook, J. In Molecular Cloning: A Laboratory Manual; Maniatis, T., Fritsh, E. F., Sambrook,
Blotechnol. Rog., 1993, Vol. 9, No. 3
J.,Eds.; Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, 1982. Masaki, T.; Tanabe, M.; Nakamura, K.; Soejima, M. Studies on aNew ProteolyticEnzymefrom Achromobacter lyticus M4971. Biochim. Biophys. Acta 1981, 660,44-50. Matauda, A,; Matauyama, K.; Yamamoto, K.; Ichikawa, S.; Komatau, K.-I. Cloning and Characterization of the Genes for Two Distinct Cephalosporin Acylases from a Pseudomonas Strain. J. Bacteriol. 1987a, 169, 5815-5820. Matauda, A,; Toma, K.; Komatau, K.-I. Nucleotide Sequences of the Genes for Two Distinct Cephalosporin Acylases from a Pseudomonas Strain. J. Bacteriol. 198713, 169, 58216826. McClure, W. R. Mechanism and Control of Transcription Initiation in Prokaryotes. Annu. Rev. Biochem. 1985,54,171204. Meister, A.; Tate, S. S.; Griffith, 0. W. y-Glutamyl Transpeptidase. Methods Enzymol. 1981, 77, 237-253. Mondello, F. J. Cloning and Expression in Escherichia coli of Pseudomonas Strain LB400 Genes Encoding Polychlorinated Biphenyl Degradation. J. Bacteriol. 1989, 171, 17251732. Nakayama, R.; Kumagai, H.; Tochikura, T. Purification and Properties of y-Glutamyltranspeptidasefrom Proteus mirabilis. J . Bacteriol. 1984a, 160, 341-346. Nakayama, R.; Kumagai, H.; Tochikura, T. Leakage of Glutathione from Bacterial Cells Caused by Inhibition of y-Glutamyltranspeptidase. Appl. Environ. Micro biol. 198413, 47,653-657. Oh, S.-J.;Kim, Y.-C.; Park, Y.-W.; Min, S.-Y.; Kim, I.-S.; Kang, H A . Complete Nucleotide Sequence of the Penicillin G Acylase Gene and the Flanking Regions, and Ita Expression in Escherichia coli. Gene 1987,56, 87-97. Papandrikopoulou, A,; Firey, A.; Gassen, H. G. Cloning and Expression of y-Glutamyl Transpeptidase from isolated porcine brain capillaries. Eur. J. Biochem. 1989, 183, 693698. Perlman, D.; Halvorson, H. 0. A Putative Signal Peptidase Recognition Site and Sequence in Eukaryotic and Prokaryotic Signal Peptides. J. Mol. Biol. 1983, 167, 391-409. Rajagopalan, S.; Park, J.-H.; Patel, P. D.; Levovitz, R. M.; Lieberman, M. W. Cloning and Analysis of the Rat y-Glutamyltransferase Gene. J. Biol. Chem. 1990,265, 11721-11725. Rajpert-De Meyta, E.; Heisterkamp, N.; Groffen, J. Cloning and Nucleotide Sequence of Human y-Glutamyl Transpeptidase. Proc. Natl. Acad. Sci. U.S.A. 1988,85, 8840-8844. Sakamuro, D.; Yamazoe, M.; Matauda, Y.; Kangawa, K.; Taniguchi, N.; Matauo, H.; Yoshikawa, H.; Ogasawara, N. The primary Structure of Human y-Glutamyl Transpeptidase. Gene 1988, 73, 1-9.
331
Sanger, F.; Nicklen, S.; Coulson, A. R. DNA Sequencing With Chain-Terminating Inhibitors. Proc. Natl. Acad. Sci. U.S.A. 1977, 74, 5463-5467. Stark, M. J. R. Multiple Expression Vectors Carring the lac Repressor Gene for Regulated High-Level Expression of Genes in Escherichia coli. Gene 1987, 51, 255-267. Suzuki, H.; Kumagai, H.; Tochikura, T. y-Glutamyltranspeptidase fromEscherichia coli K-12: Purification and Properties. J. Bacteriol. 1986a, 168, 1325-1331. Suzuki, H.; Kumagai, H.; Tochikura, T. y-Glutamyltranspeptidase from Escherichia coli K-12: Formation and Localization. J. Bacterid. 198613, 168, 1332-1335. Suzuki, H.; Kumagai, H.; Echigo, T.; Tochikura, T. DNA Sequence of the Escherichia coli K-12 y-Glutamyltranspeptidase Gene, ggt. J. Bacteriol. 1989, 171, 5169-5172. Takeshita, S.; Sato, M.; Toba, M.; Masahashi, W.; HashimotoGotoh, T. High-Copy-Number and Low-Copy-Number Plasmid Vectors for lac2 a-Complimentation and Chloramphenicol- or Kanamycin-resistance Selection. Gene 1987, 61, 6374. Tate, S. S.; Meister, A. Affinity Labeling of y-Glutamyl Transpep tidase and Location of the y-Glutamyl Binding Site on the Light Subunit. Proc. Natl. Acad. Sci. U.S.A. 1977, 74,931935. Tate, S. S.; Ross, M. E. Human Kidney y-Glutamyl Transpeptidase. J. Biol. Chem. 1977, 252, 6042-6045. Tate, S. S.; Meister, A. y-Glutamyl Transpeptidase: catalytic, structural and functional aspects. Mol. Cell. Biochem. 1981, 39,357-368. Tate, S. S.; Meister, A. y-Glutamyl Transpeptidase from Kidney. Methods Enzymol. 1985,113,400-419. Tate, S. S.; Khadse, V.; Wellner, D. Renal y-Glutamyl Transpeptidase: Structural and ImmunologicalStudies. Arch. Biochem. Biophys. 1988,262, 397-408. Towbin, H.; Staehelin, T.; Gordon, J. Electrophoretic Transfer of Proteins from Polyacrylamide Gels to Nitrocellulose Sheets: Procedure and Some Applications. Proc. Natl. Acad. Sci. U.S.A. 1979, 76, 4350-4354. von Hippel, P. H.; Bear, D. G.; Morgan, W. G.; Mcswiggen, J. Protein-Nucleic Acid Interactions in Transcription: A Molecular Analysis. Annu. Rev. Biochem. 1984, 53, 389446. Yanisch-Perron, C.; Viera, J.; Messing, J. Improved M13 Phage Cloning Vectors and Host Strain: Nucleotide Sequences of the M13mp18 and pUC19 Vectors. Gene 1985, 33, 103109. Zylstra, G. J.; Gibson, D. T. Toluene Degradation by Pseudomonas putida F1. J . Biol. Chem. 1989,264, 14940-14946. Accepted February 17, 1993.
