J. Biochem. 118, 555-561 (1995)

Cloning and Expression of from Acinetobacter calcoaceticus ATCC 230551

Hideki Adachi2 and Masafumi Tsujimoto

Suntory Institute for Biomedical Research, Shimamoto, Mishima-gun, Osaka 618

Received for publication, April 3, 1995

The gene encoding dipeptidase was cloned from Acinetobacter calcoaceticus ATCC 23055 . D etermination of the nucleotide sequence revealed that the gene had an open reading flame of 1,050 by coding a protein of 350 amino acids. The deduced amino acid sequence showed 48.8% similarity to human renal dipeptidase and conserved two amino acid residues identified in human and pig renal as essential ones for the catalytic activity . Purified recombinant expressed in Escherichia coli did not hydrolyze the unsatu rated dipeptide, glycyldehydrophenylalanine. On the other hand , it preferentially hydrol yzed dipeptides having a D-amino acid, when compared with those having an L-amino acid at the C-terminal. Furthermore, it could not hydrolyze tripeptides . These results indicate that the dipeptidase produced by A. calcoaceticus ATCC 23055 has a unique substrate specificity and preferentially hydrolyzes dipeptides having a D-amino acid at the C-termi nal.

Key words: Acinetobacter calcoaceticus, D-amino acid, ancestral gene, Co2i, renal dipep tidase.

Renal dipeptidase (rDP) is a zinc-containing ectoenzyme mammalian ƒÀ-lactamase (15), there is a possibility that the which hydrolyzes a wide range of dipeptides (1). rDP exists gene product of R is a ƒÀ-lactamase in A. calcoaceticus. as a disulfide-linked homo-dimeric glycoprotein of subunit However, no definitive data are available on the complete M, 59,000 in human (2). The enzyme has been implicated nucleotide sequence or the physiological role of the prod in the renal of glutathione and its conjugates ucts from R and orfX. (e.g. D,) and is responsible for the hydrolysis of In this paper, we have cloned for the first time the gene f3-lactam antibiotics such as penem and carbapenem deriv acdp, encoding dipeptidase, from the prokaryote A. cal atives (2-5). coaceticus ATCC 23055, and sequenced it completely. We Recently we and others cloned cDNAs encoding the found that the encoded protein (ACDP) conserved essential enzyme from several mammalian species and found that amino acid residues identified in mammalian dipeptidases. these showed extensive similarity to each other In spite of the structural similarity between ACDP and (6-9). Availability of cDNA of the enzyme and recom mammalian dipeptidases, ACDP could not hydrolyze the binant protein made it possible to characterize the enzyme, unsaturated dipeptide glycyldehydrophenylalanine. Un and amino acid residues essential for the catalytic activity expectedly, analysis of substrate specificity toward various were identified (10, 11). dipeptides employing recombinant enzyme showed that the On the other hand, it has been suggested that in some enzyme preferentially hydrolyzed dipeptides containing a prokaryotic cells such as Acinetobacter calcoaceticus and D-amino acid at the C-terminal, in sharp contrast with Klebsiella pneumoniae, a gene which shows significant mammalian dipeptidase. homology to mammalian dipeptidase is coded near the pqq genes [genes involved in pyrrolo-quinoline-quinone (PQQ) MATERIALS ANDMETHODS biosynthesis] (12, 13). They are designated as R and orfX, respectively. The genus Acinetobacter is intrinsically resis Materials-A. calcoaceticus ATCC 23055 was obtained tant to ƒÀ-lactam antibiotics and the intrinsic resistance to from the American Type Culture Collection. Recombinant ƒÀ-lactam antibiotics is attributed to the production of human renal dipeptidase was expressed in CHO cells and ƒÀ-lactamase (14). Since it was reported that rDP is a purified as described previously (9). Glycyldehydrophenyl alanine (GDPA) was synthesized as described by Campbell 1 The nucleotide sequence data reported in this paper will appear in (16). Dipeptides were obtained from Sigma, Tokyo Kasei the GSDB, DDBJ, EMBL, and NCBI nucleotide sequence databases Kogyo and Peptide Institute. L-Amino acid oxidase from with the accession number D50330. Crotalus durissus and D-amino acid oxidase from porcine 2 To whom correspondence should be addressed . Tel: 181-75-962 kidney were purchased from Boehringer Mannheim and 9283, Fax: +81-75.962-6448 Sigma, respectively. Peroxidase was obtained from Wako Abbreviations: ACDP, Acinetobacter calcoaceticus dipeptidase; Pure Chemicals. CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonic acid; GDPA, glycyldehydrophenylalanine; PQQ, pyrrolo-quinoline Construction of Genomic DNA Library from A. calcoace quinone; rDP, renal dipeptidase. ticus ATCC 23055-Chromosomal DNA was obtained

Vol. 118, No. 3, 1995 555 556 H. Adachi and M. Tsujimoto

from a culture of A. calcoaceticus ATCC 23055 by employ mM Tris/HC1 pH 7.6 containing 0.1% CHAPS) and then ing an IsoQuick DNA Extraction Kit (Micro Probe). In our applied to a DEAE-Sepharose Fast Flow (Pharmacia) initial experiment, a Southern blot analysis was conducted column previously equilibrated with buffer A. Proteins using chromosomal DNA from A. calcoaceticus ATCC were eluted with a linear gradient of NaCl (0-500 mM) in 23055 and PCR product prepared as described below. It buffer A. The active fractions were pooled and applied to a revealed that an approximately 6 or 3 kbp fragment of Zn2+-Chelating Sepharose (Pharmacia) column previously chromosomal DNA was hybridized with the probe when it equilibrated with buffer A. Proteins were eluted with a was digested with Sall and BglII or SacI and EcoT22I. The linear gradient of glycine (0-500 mM) in buffer A. The chromosomal DNA was digested with SacI and BglII or active fractions were then pooled and applied to a hydroxy SacI and EcoT22I, and fractionated by 1.2% agarose gel apatite (Seikagaku Kogyo) column previously equilibrat electrophoresis, DNA fragments of approximately 6 or 3 ed with buffer A. Proteins were eluted with a linear kbp were purified using Gene Clean II (BIO 101). DNA gradient of KH2PO4 (0-300 mM) in buffer A. After addition fragments were ligated into pUC19 and the 'ligation of (NH,)2SO, to 30% saturation, the active fractions were mixture was transfected into ElectroMAX DH10B cells applied to a Butyl-Sepharose Fast Flow (Pharmacia) col (GIBCO BRL). After titration of the library on Nutrient umn previously equilibrated with 50 mM Tris/HC1 pH 7.6 agar plates containing ampicillin (50 pg/ml), the rest of the containing 30% (NH,)2SO4. Proteins were eluted with a library was plated on 96-well plates with 200 clones per linear gradient of (NH,),SO, (30-0%) in 50 mM Tris/HC1 well. In total, 112,000 and 209,000 clones were obtained, pH 7.6. respectively. Protein concentration was determined according to the Screening of acdp from Genomic Library-Two oligonu method described by Bradford (20). cleotides, 5•L-ATGAAGCCCCGCCATATTCCAGTT-3•L and Enzyme Assay-Dipeptidase activity towards GDPA was 5•L CAAAAAAGCTTTCTCATTCATATG-3•L were synthe measured as the rate of decrease in absorbance at 274 nm, sized based on the nucleotide sequence of R gene of A. as described (16). The activities against various dipeptides calcoaceticus (12). A genomic library was screened em and tripeptides were determined essentially according to ploying these primers as described (17, 18). In brief, the Sugiura et al. (21). The reaction was initiated by the genomic library was distributed on 96-well plates with 200 addition of 25ƒÊ1 of enzyme to 250ƒÊ1 of 4 mM dipeptide clones per well, and the supernatants were pooled in every solutions in 50 mM Tris/HCl pH 7.6 and 500ƒÊl of coloring column and row. PCR was carried out with these primers to reagent containing 1.5 units of peroxidase, 0.05ƒÊl of fi nd wells containing DNA for acdp. The positive pools were N,N-dimethylaniline, 0.02 mg of 4-aminoantipyrine, and plated, and every colony was checked by PCR. DNAs of the 0.1 unit of L-amino acid oxidase or 0.2 unit of D-amino acid positive clones were recloned into M13mp19 vector in both oxidase and 1 mM CoCl2 in 50 mM Tris/HC1 pH 7.6. After orientations and sequenced using Taq Dye DeoxyTM Ter incubation for 20 to 40 min at 37•Ž, reaction was terminat minator Cycle Sequencing Kit and an Applied Biosystems ed by the addition of 0.25ml of 0.1 M acetic acid, then the model 373A DNA Sequencer. DNAs of the positive clones absorbance at 550nm was measured. One unit of the were deleted using a double-stranded nested deletion kit activity was defined as the amount of the enzyme which (Pharmacia) and then sequenced. The nucleotides and hydrolyzed one micromole of substrate per minute under deduced amino acid sequence were analyzed with the the assay conditions. The kinetic parameters were obtained Sequence Analysis Package by Genetics Computer (the from a Lineweaver-Burk plot using 0.64-515mM Leu GCG Package). D-Leu as a substrate. The effects of various inhibitors were Expression of ACDP in E. coli-Two oligonucleotides, tested in the presence of 1 mM inhibitor. The activity 5•L-GTAGAATTCATGAAGCCTAGCCATATTCC-3•L and 5•L-CAGGTCGACTTATTCACCCAGAATCCGAT-3•L, were synthesized based on the nucleotide sequence of acdp and used as PCR primers. PCR was carried out using genomic DNA of A. calcoaceticus as a template. The obtained PCR fragment was digested with EcoRI and Sail and ligated into the expression vector pUC-PL-cI (19). The ligation mix tures were transfected into E. coli W31 10 and the transfor mants were cloned. The nucleotide sequences of these clones were determined after recloning into the M13mp19 vector in both orientations. Among these transformants E. coli W3110/pUC-PL-cI ACDP-4 was used for further characterization. The recombinant protein for ACDP was expressed as described (19). Briefly, the transformant was cultured in Terrific broth at 32•Ž. At the late log phase, the temperature was shifted to 42•Ž, and the culture was continued for 3 h. The cells were then collected by centri fugation at 5,000•~g for 10 min, washed twice with phos phate-buffered saline, and resuspended in the same buffer. The cells were disrupted by sonication and centrifuged at Fig. 1. Partial restriction map and sequencing strategy of genomic clones containing acdp gene (Acinetobacter calcoace 5,000•~g for 10 min, and the supernatant was collected. ticus dipeptidase gene). The regions and sizes of the cloned Purification of ACDP from E. coli W3110 pUC-P, -cI fragments are indicated. The open reading frames are indicated by ACDP-4-The cell lysate was dialyzed against buffer A (50 open bars.

J. Biochem. Cloning and Expression of A . calcoaceticus Dipeptidase 557

against Lys-Lys was determined according to Shimura and addition of 12.5ƒÊl of enzyme to 3ml of 50mM Tris/HCl Vogel (22). In brief , the reaction was initiated by the pH 7.6 containing 2mM Lys-Lys and 1mM CoCl2 and

Fig. 2. Nucleotide and de duced amino acid sequences of acdp. The initiation codon (287 -289) for acdp is underlined. The partial C-terminal sequence of PQQIII gene locating upstream of acdp is also shown.

Vol. 118, No. 3, 1995 558 H. Adachi and M. Tsujimoto

incubated for 20 to 40 min at 37•Ž. Then the reaction was library on 96-well plates, PCR was carried out using terminated by heating the reaction mixture in a boiling primers described in "MATERIALS ANDMETHODS" to find water bath for 10 min. To each tube, 0.2 ml of 3 M HCl and the wells containing the DNA for acdp. Positive wells were 0.5 ml of 15% (w/v) ninhydrin in 2-methoxyethanol were then plated and every colony was again checked by PCR. added and the reaction mixture was heated in a boiling One clone, AC12, containing an insert of approximately 3 water bath for 60 min. It was cooled to room temperature, kbp of A. calcoaceticus chromosomal DNA was obtained. then 4 ml of 15 M phosphoric acid was added and the The nucleotide sequence of the clone was determined and absorbance at 515 nm was measured. we found that it lacked 45 by of the N-terminal region of Metal Content of ACDP-The enzyme (0.07mg/ml) was the gene designated as R or orfX. Then we constructed analyzed for zinc (213.9nm) using a Hitachi Z-8200 another library and cloned AC8 after digestion of the

polarized Zeeman atomic absorption spectrophotometer. chromosomal DNA with BglII and Sacl. Sequence analysis Protein concentration was determined according to the showed that AC8 contained the coding region of the R gene reported method (10). In brief, after gas-phase hydrolysis lacking in AC12 and had an overlapping sequence with it of the sample by HC1 at 150•Ž for 1 h under reduced (Fig. 1). pressure, amino acid derivatives were separated by re Sequence Analysis of Dipeptidase Gene-Figure Z shows verse-phase HPLC using a PTC column (4.5•~200mm, the complete nucleotide sequence and deduced amino acid Wako) after treatment with phenyl isothiocyanate (23). sequence of A. calcoaceticus dipeptidase (ACDP). It was found that 18 by downstream of the stop codon for PQQIII RESULTS gene, there was an open reading frame of 1,050 bp coding for a polypeptide of 350 amino acids with a calculated Cloning of Dipeptidase Gene from A. calcoaceticus molecular weight of 39.4 kDa. The deduced amino acid ATCC 23055-Total DNA from A. calcoaceticus ATCC sequence of this protein has 48.8% similarity to human 23055 was digested with SacI and EcoT22I, size fractionat renal dipeptidase. Essential amino acid residues for cata ed and ligated into pUC19. The ligation mixture was lytic activity identified in mammalian renal dipeptidases transfected into E. coli DH10B. After distribution of the (i.e. Glu125 for human and His219 for pig renal dipeptidase) were conserved (see below). It should be noted that the nucleotide and deduced amino acid sequences differ slightly from the published ones (e.g. the N-terminal 290 amino acids of ACDP show 86.9% identity to 290 amino acids of ORF R) (12). These results presumably reflect the differ ence in the strain employed. Expression of Dipeptidase in E. coli-The coding region of ACDP was ligated into pUC-PL-CI, transfected into E. coli W3110 and cloned. The 10,000•~g supernatants of the lysates of the clone and parent cells which were grown at 42•Ž were collected. The 40 kDa band which was expected from the calculated molecular weight of ACDP was detect ed only in the supernatant of the transformant, but not that of the parent cell (Fig. 3B). However, we could not detect

TABLE II. Inhibition of ACDP purified from E. coli W3110/ pUC-PL-cI ACDP 4. Purified enzyme was assayed in the presence or absence of various inhibitors at 1 mM using Leu-D-Leu under the conditions described in "MATERIALS and METHODS." Fig. 3. Hydrolysis of Leu-D-Leu by recombinant ACDP ex pressed in E. coli. A: Cells were transfected with or without pUC PL-cI ACDP-4 and then cloned. After sonication, cell lysate was collected and dipeptidase activity was assayed employing Leu-D-Leu as a substrate. B: SDS-polyacrylamide (12.5%) gel electrophoresis of standard proteins (Pharmacia) (lane 1), cell lysate containing recom binant ACDP (lane 2), and purified recombinant ACDP (lane 3). The molecular weights (kDa) of standard proteins are shown in the figure.

TABLE I. Purification of ACDP from E. coli W3110/pUC-PL-cI ACDP 4. All fractions were assayed using Leu-D-Leu or Leu-Leu under the conditions described in "MATERIALS AND METHODS."

J. Biochem. Cloning and Expression of A. calcoaceticus Dipeptidase 559 any hydrolytic activity when glycyldehydrophenylalanine the Leu-D-Leu and Leu-Leu hydrolytic activities during the (GDPA), a typical substrate for renal dipeptidase (16), was purification procedure. Table I presents a summary of the employed as a substrate. Moreover, we could not detect any purification procedures, showing that the enzyme was difference in hydrolytic activities between the supernatants purified about 27.9-fold with a recovery of 29.4%. The final of the transformant and parent cells using Leu-Leu as a preparation showed a single band at about 40 kDa on substrate. On the other hand, when Leu-D-Leu was em SDS-PAGE analysis (fig. 3B, lane 3) and the Km and Vmax ployed, we observed apparent hydrolytic activity only in values toward Leu-D-Leu measured in the presence of 1 the supernatant of the transformant (Fig. 3A). These mM Co2+ were determined to be 9.3mM and 934nmol/ results suggested that ACDP showed the dipeptidase min/mg, respectively. activity toward Leu-D-Leu, but the substrate specificity When enzyme activity was assayed in the presence of 1 might be different from those of mammalian renal dipep mM inhibitors, the enzyme was not inhibited markedly by tidases. any reagent tested (Table II). Bestatin (an inhibitor of Purification and Characterization of Recombinant ), EDTA and o-phenanthroline (chelators ACDP-To examine the substrate specificity of ACDP of metal ions) and p-chloromercuriphenylsulfonic acid more exactly, we purified the recombinant enzyme from E. (which attacks sulfhydryl residues of protein) inhibited the coli W3110/pUC-PL-cI ACDP-4 lysate. To separate the enzyme slightly. These results indicated that some metal recombinant enzyme activity from intrinsic Leu-Leu ion(s) and sulfhydryl residue(s) might play a role in the hydrolyzing activity of E. coli W3110 lysate, we followed enzyme activity. The effect of other reagents such as DTT,

Fig. 4. Effect of divalent metal ions on the hydrolytic activity of ACDP. The enzyme activity was measured employing Leu-D-Leu as a substrate in the presence (1mM) or absence of metal ions. The enzyme activity was also measured in the presence of 1 mM EDTA.

TABLE III. Substrate specificity of ACDP. Purified enzyme was assayed using various peptides under the conditions described in "MATERIALS and METHODS ."

Fig. 5. Preferential hydrolysis of dipeptides having a D-amino acid at the C-terminal by ACDP. A: Dipeptides were incubated with recombinant ACDP (123ƒÊg/ml 25ƒÊ1) and the enzyme activity was measured as described in "MATERIALS AND METHODS." B: Dipeptides were incubated with recombinant ACDP (123ƒÊg/ml 25 ƒÊl, closed bar) or recombinant human rDP (1.7 ng/ml 25ƒÊl, open bar). Relative hydrolytic activity is shown in the figure using Leu-Leu hydrolyzing activity as a standard. The specific activities of ACDP and recombinant human rDP toward Leu-Leu were 5.2 and 123,196 nmol/min/mg respectively.

Vol. 118, No. 3, 1995 560 H. Adachi and M. Tsujimoto

PMSF, and cysteinyl-bis-glycine were not determined , b activity could be detected (Fig. 5A). These results suggest ecause these reagents interfered with quantitive analysis ed that there is a stereospecific interaction between enzyme of the amino acid released from the dipeptide. and substrates, which is required for the activity. The Figure 4 shows the effect of various divalent metal ions importance of the D-configuration at the C-terminal was on the hydrolytic activity toward Leu-D-Leu. In the absence also observed with a different set of substrates (fig. 5B). In of exogenously added metal ions, a specific activity of 7.7 these cases, the enzyme hydrolyzed substrates having a nmol/min/mg was obtained. In the presence of EDTA , a D-amino acid at the C-terminal. On the other hand, recom decrease in the enzyme activity was observed, suggesting binant human rDP expressed in CHO cells showed relative that some metal ions were required for maximum activity . ly equal activities toward these substrates, suggesting that Addition of metal ions such as Zn2+, Cot+, and Ni2+ in interaction between substrate and enzyme was less stereo creased the activity, while Ca2+ or Mn2+ showed little effect specific than that of ACDP. on the activity. The most effective ion tested was Co2+

(specific activity of 91.9 nmol/min/mg). DISCUSSION To confirm that the enzyme was indeed the metallopep tidase, we analyzed its Zn content. We found that the In this study, we have cloned and expressed in E. coli the enzyme contained 1.2 mol of zinc per molecule of the gene coding for dipeptidase activity from A. calcoaceticus enzyme. ATCC 23055. As reported by Goosen et al. (12), A. Table III summarizes the substrate specificity of ACDP. calcoaceticus dipeptidase (ACDP) gene was located just Among substrates tested, Gly-D-Ala and Gly-D-Leu were downstream of the gene for PQQIII. The acdp gene had an hydrolyzed most efficiently. However, the enzyme did not open reading frame of 1,050 by coding for a polypeptide of hydrolyze any tripeptide tested. When the carboxy termi 350 amino acids with a calculated weight of 39.4 kDa. nal of Leu-Leu was modified, hydrolytic activity of the Recombinant protein expressed in E. coli showed a molecu enzyme could no longer be detected. Furthermore, we could lar weight of 40 kDa on SDS-PAGE analysis and was not detect D-aminoacylase activity or aminopeptidase preferentially active toward dipeptides having a D-amino activity. These results indicated that the enzyme indeed acid at the C-terminal, indicating that the enzyme showed had dipeptidase activity, but its substrate specificity was apparent stereospecificity toward the substrates. These different from that of mammalian dipeptidases. results suggest that stereospecific interaction between Figure 5 shows the hydrolytic activity of ACDP toward enzyme and substrate was required for the activity. different dipeptides. When hydrolytic activities against the Mammalian rDPs are zinc metallopeptidase containing 1 four enantiomers of Leu-Leu (i.e. Leu-Leu, Leu-D-Leu, mol of Zn2+ per subunit (1), and we have found in this study D-Leu-Leu, and D-Leu-D-Leu) were compared, it was that ACDP also contained 1.2 mol Zn2+ per mol of the evident that Leu-D-Leu was preferentially hydrolyzed. enzyme. However, because the addition of metal ions such When the N-terminal of substrates was an L-amino acid, as Zn2+, Cot+, and Ni2+ increased the catalytic activity of some hydrolyzing activity was detected, whatever the the enzyme, this enzyme might require two or more metal configuration at the C-terminal. However, if the N-termi ions for its maximum activity. It should be noted here that nal amino acid was of D-configuration, no hydrolyzing among the metal ions tested, Co2+ was most effective in

Fig. 6. Alignment of the predicted amino acid sequences of indicated by dashes. Amino acid residues conserved among all ACDP and mammalian renal dipeptidases. Accession number in dipeptidases are boxed. The arrows indicate the essential amino acid the GSDB/DDBJ/EMBL/NCBI DNA databases are given in parenthe residues so far identified for the catalytic activity of the enzyme. sis. Alignment was obtained by using the PILEUP program. Gaps are

J. Biochem. Cloning and Expression of A . calcoaceticus Dipeptidase 561 enhancing the enzyme activity , and a Col-dependent di peptidase. Biochem. J. 280, 71-78 peptidase has been reported in E. coli (24). However , ACDP 9. Adachi, H., Ishida, N., and Tsujimoto, M. (1992) Primary should be different from the Co2+-dependent dipep structure of rat renal dipeptidase and expression of its mRNA in tidase in E. coli, because it could not hydrolyze Lys-Lys rat tissues and COS-1 cells. Biochim. Biophys. Acta 1132, 311 - 314 efficiently (16.4nmol/min/mg) . 10. Adachi, H., Katayama, T., Nakazato, H., and Tsujimoto, M. Computer search revealed that the amino acid sequence (1993) Importance of Glu-125 in the catalytic activity of human of ACDP has significant homology only to mammalian rDP renal dipeptidase. Biochim. Biophys. Acta 1163, 42-48 (Fig. 6). While human rDP shows 83.4% similarity with rat 11. Keynan, S., Hooper, N.M., and Turner, A.J. (1994) Directed rDP, 48.8% similarity was observed between human rDP mutagenesis of pig renal : His219is critical and ACDP. However, it was reported that rDP is a but the DHXXH motif is not essential for zinc binding or catalytic activity. FEBS Lett. 349, 50-54 mammalian ƒÀ-lactamase (15), ACDP has no similarity to 12. Goosen, N., Horsman, H.P.A., Huinen, R.G.M., and van de metallo-ƒÀ-lactamases and Zn2+-containing D-alanyl-D-ala Putte, P. (1989) Acinetobacter calcoaceticus genes involved in nine peptidases. biosynthesis of the coenzyme pyrrolo-quinoline-quinone: Nu At least two amino acid residues are essential for the cleotide sequence and expression in Escherichia coli K-12. J. hydrolytic activity of the enzymes (10, 11). These two Bacteriol. 171, 447-455 amino acid residues (i. e. Glu125and His219in human rDP and 13. Meulenberg, J.J.M., Sellink, E., Riegmn, N.H., and Postma, porcine rDP) were alssssssconserved in ACDP. These results P.W. (1992) Nucleotide sequence and structure of the Klebsiella suggest, although further characterization is required, that pneumoniae pqq operon. Mol. Gen. Genet. 232, 284-294 14. Joly-Guillou, M.L., Bergogne-Berezin, E., and Moreau, N. these genes developed from a common ancestral gene. (1987) Enzymatic resistance to ƒÀ-lactams and aminoglycosides in In summary, we have cloned the gene for dipeptidase Acinetobacter calcoaceticus. J. Antimicrob. Chemother. 20, 773 from A. calcoaceticus ATCC 23055 and determined the - 776 complete nucleotide sequence. This is the first dipeptidase 15. Campbell, B.J., Forrester, L.J., Zahler, W.L., and Burks, M. characterized from a prokaryotic cell. The enzyme shows (1984) ƒÀ-Lactamase activity of purified and partially character unique properties of substrate specificity and metal ion ized human renal dipeptidase. J. Biol. Chem. 259, 14586-14590 16. Campbell, B.J. (1970) Renal dipeptidase in Methods in En requirement. The availability of recombinant protein will zymology (Perlmann, G.E. and Lorand, L., eds.) Vol. 19, pp. 722 make it possible to characterize the enzyme in detail. - 729, Academic Press, New York 17. Kwiatkowski, T.J., Jr., Zoghbi, H.Y., Ledbetter, S.A., Ellison, K.A., and Chinault, A.C. (1990) Rapid identification of yeast REFERENCES artificial chromosome clones by matrix pooling and crude lysate 1. Armstrong, D.J., Mukhopadhyay, S.K., and Campbell, B.J. PCR. Nucleic Acids Res. 18, 7191-7192 18. Isola, N.R., Harn, H.J., and Cooper, D.L. (1991) Screening (1974) Physicochemical characterization of renal dipeptidase. J. recombinant DNA libraries: A rapid and efficient method for Biol. Chem. 13,1745-1750 isolating cDNA clones utilizing the PCR. BioTechniques 11, 580 2. Adachi, H., Kubota, I., Okamura, N., Iwata, H., Tsujimoto, M., - 582 Nakazato, H., Nishihara, T., and Noguchi, T. (1989) Purification 19. Kuroki, M., Murakami, M., Wakisaka, M., Ikeda, S., Oikawa, S., and characterization of human microsomal dipeptidase. J. Biochem. 105, 957-961 Oshima, T., Nakazato, H., Kosaki, G., and Matsuoka, Y. (1992) Immunoreactivity of recombinant carcinoembryonic antigen 3. Kozak, E.M. and Tate, S.S. (1982) Glutathione-degrading enzymes of microvillus membranes. J. Biol. Chem. 257, 6322 proteins expressed in Escherichia coli. Immunol. Invest. 21, 241 - 257 - 6327 20. Bradford, M.M. (1976) A rapid and sensitive method for the 4. Hirota, T., Nishikawa, Y., Tanaka, M., Igarashi, T., and Kita quantitation of microgram quantities of protein utilizing the gawa, H. (1986) Characterization of dehydropeptidase I in the rat lung. Eur. J. Biochem. 160, 521-525 principle of protein-dye binding. Anal. Biochem. 72, 248-254 21. Sugiura, M., Ito, M., Hirano, K., Sasaki, M., and Sawaki, S. 5. Campbell, B.J., Shih, Y.D., Forrester, L.J., and Zahler, W.L. (1977) A new method for peptidase activity measurement in (1988) Specificity and inhibition studies of human renal dipep serum and tissues, using L-Leu-L-Leu as substrate. Clin. Chim. tidase. Biochim. Biophys. Acta 956, 110-118 Acta 78, 381-389 6. Adachi, H., Tawaragi, Y., Inuzuka, C., Kubota, I., Tsujimoto, M., 22. Shimura, Y. and Vogel, H.J. (1966) Diaminopimelate decarbox Nishihara, T., and Nakazato, H. (1990) Primary structure of ylase of Lemna perpusilla. Partial purification and some prop human microsomal dipeptidase deduced from molecular cloning. erties. Biochim. Biophys. Acta 118, 396-404 J. Biol. Chem. 265, 3992-3995 23. Takahashi, T., Ikai, A., and Takahashi, K. (1989) Purification 7. Rached, E., Hooper, N.M., James, P., Semenza, G., Turner, A., and characterization of proline-ƒÀ-naphthylamidase, a novel and Mantei, N. (1990) cDNA cloning and expression in Xenopus enzyme from pig intestinal mucosa. J. Biol. Chem. 264, 11565 laevis oocyte of pig renal dipeptidase, a glycosyl-phosphatidyl -11571 inositol-anchored ectoenzyme. Biochem. J. 271, 755-760 24. Ota, A. (1993) A pH and Co2+-dependent dipeptidase from 8. Igarashi, P. and Karniski, L.P. (1991) Cloning of cDNAs encoding Escherichia coli. Microbios 75, 33-38 a rabbit renal brush border membrane protein immunologically related to band 3. Sequence similarity with microsomal di

Vol. 118, No. 3, 1995