J. Biochem. 116, 818-825 (1994)

Isolation and Characterization of the Prolyl Gene (pap) from Aeromonas sobria: Comparison with the Bacillus coagulans Enzyme1

Ana Kitazono,* Atsuko Kitano,* Daisuke Tsuru,•õ and Tadashi Yoshimoto*,2

*School of Pharmaceutical Sciences , Nagasaki University, 1-14 Bunkyo-machi, Nagasaki, Nagasaki 852; and •õ Department of Applied Microbiology, Kumamoto Institute of Technology, 4-22-1 Ikeda, Kumamoto, Kumamoto 860

Received for publication, May 16, 1994

The Aeromonas sobria pap gene encoding prolyl aminopeptidase (PAP) was cloned. It consists of 425 codons and encodes a homotetrameric of 205kDa. The purified enzyme showed an almost absolute specificity for amino-terminal . Proline and hydroxyproline residues from many peptide and amide substrates could be easily removed, while no activity was detected for substrates having other amino terminals. The enzyme was very similar to that from Bacillus coagulans in many aspects, such as the strong inhibition caused by PCMB and the weak or no inhibition caused by DFP and chelators, respectively. However, these show only 15% identity in their amino acid sequences. Differences were also observed in their molecular weight, stability and activity toward some peptide substrates. When aligning the deduced amino acid sequence with known sequences from other microorganisms, conserved sequences were found at the amino-terminal region; the significance of these conserved regions is discussed. Based on the results of this work, and on the studies available to date, the occurrence of at least two types of PAPs is postulated. One group would be formed by the Bacillus, Neisseria, and Lactobacillus enzymes, and the other by enzymes such as the Aeromonas PAP.

Key words: Aeromonas sobria, aminopeptidase, iminopeptidase, proline.

Prolyl aminopeptidase (PAP, proline aminopeptidase, Pro inhibitor, 3,4-dichloroisocoumarin, although no inhibition X aminopeptidase, proline iminopeptidase) [EC 3.4.11.5] by DFP or by any other related chemical was reported (6). catalyzes the removal of amino-terminal proline from On the other hand, Atlan et al. found high similarities peptides, and has been the object of many studies since the among the amino acid sequences of the Bacillus and first report by Sarid et al. of the Escherichia coli enzyme Lactobacillus PAP enzymes and a group of from (1, 2). Different sources of the enzyme have been described Pseudomonas putida (5). The significance of this finding has implying its wide distribution in nature. However, reports not yet been discussed, and it is possible that these enzymes concerning microorganisms and plants are the most abun bear both and aminopeptidase activities, as has dant, and attempts to isolate the enzyme from mammalian been demonstrated for leukotriene A4 hydrolase (LTA, tissues have so far failed, suggesting the absence of a true hydrolase) [EC 3.3.2.5]. The predicted zinc of PAP activity in the latter sources. this enzyme had been found to have a striking similarity to To date, there are three pap genes with known primary the corresponding primary structures of certain aminopep structures: the Bacillus coagulans (3), Neisseria gonor tidases and neutral . Later work revealed that the rhoeae (4), and Lactobacillus delbrueckii subsp. bulgaricus LTA, hydrolase (which is also an epoxide hydrolase whose (5) genes. Homologous regions at the amino terminal were is a fatty acid), also possesses a significant found by aligning the deduced amino acid sequences. The peptidase activity functionally related to aminopeptidase identities range from 23% for the Bacillus and Neisseria M. The studies led to the conclusion that LTA, is a Zn2+ enzymes to 36% for the Lactobacillus and Bacillus en - metallohydrolase and a bifunctional enzyme with intrinsic zymes. These enzymes share similar characteristics with aminopeptidase activity (7, 8). The enzyme was also found regard to substrate specificity, molecular weight, and to be inhibited by bestatin (9), and by captopril (10). inhibition by sulfhydryl reagents. However, the enzyme If the PAP activity happens to be also a bifunctional from L. bulgaricus was also inhibited by a serine enzyme, the difficulties in isolating and identifying a true PAP in mammalian tissues would become understandable.

1 This work was supported in part by Grants-in-Aid for Scientific Matsushima et al. reported the identity of the PAP they Research from the Ministry of Education, Science and Culture of had purified with (11), and later the Japan. A. Kitazono is a recipient of a scholarship from the same same group characterized a proline ƒÀ-naphthylamidase ministry and expresses her gratitude. from porcine intestinal mucosa, describing it as a serine 2 To whom correspondence should be addressed . hydrolase, composed of three identical subunits each with a Abbreviations: Hyp-ƒÀNA, hydroxyproline ƒÀ-naphthylamide; LAP, leucyl aminopeptidase; PAP, prolyl aminopeptidase; Pro-ƒÀNA, molecular weight of 58,000 (12). However, this enzyme

proline ƒÀ-naphthylamide; Xaa, any amino acid. was finally identified as a carbaxylesterase from studies of

818 J. Biochem. Prolyl Aminopeptidase Gene from Aeromonas sobria 819

amino acid (13). This report has 0.1% potassium phosphate dibasic), with vigorous agita recently been revised by Heymann and Peter, who tried to tion. Chromosomal DNA was prepared following the specify which of the at least two carboxylesterase isoen method of Saito and Miura (19); plasmid pBR322 was used zymes present in pig liver was identical with proline as the vector and E. coli DH1 as the host. For selection, ƒÀ-naphthylamidase. They concluded that the hydrolysis of either ampicillin (50ƒÊg/ml) or tetracycline (25ƒÊg/ml) proline ƒÀ-naphthylamide (Pro ƒÀNA) was just a minor side was added to the medium. Individual colonies of DH1 cells activity of the amidase-type isoenzyme of pig liver carbox transformed with the pBR322 plasmids from the genomic

ylesterase, and that the sequence reported by Matsushima library were picked from plates and transferred into the et al. could possibly be that of the main carboxylesterase wells of microtiter plates containing 180ƒÊl of broth. After isoenzyme of pig liver and not that of proline ƒÀ-naphthyl. overnight incubation, 50ƒÊl of the culture was transferred amidase or the amide-cleaving isoenzyme of carboxyl to another plate and mixed with 50ƒÊl of 2mM Pro-ƒÀNA. (14). So, the presence of a PAP-like enzyme in The reaction was conducted overnight at 30•Ž and stopped mammalian tissues might require reconsideration in this by adding 50ƒÊl of the Fast Garnet GBC salt solution (1mg/ still speculative new context, as is the case for the PAPs as ml in 1M acetate buffer pH 4.0 containing 10% Triton a whole. X-100). Rapid red color development indicated the pres Looking for new sources of the enzyme, we screened soil ence of high enzymatic activity. microorganisms for PAP producers, isolating a strain with DNA Manipulation, and Subcloning-Plasmid DNA was high activity. On the basis of its biochemical, cultural, and isolated by the alkaline extraction procedure (20) or by morphological characteristics, this strain was identified as CsCl-ethidium bromide equilibrium density gradient cen Aeromonas sobria. Aeromonas is a genus from which many trifugation. Competent cells for transformation were other peptidases have been isolated: the well-known leucyl prepared by rubidium chloride treatment. All other proce aminopeptidase (15), and a neutral protease from A. dures were done following the standard procedures (21). The fragment containing the pap gene was subcloned based proteolytica (16, 17). Recently the cloning of the A. hydrophila prolyl gene has also beennn report on the map constructed, and deletion ed (18). In this study, we describe the cloning, sequencing, analysis of these subclones indicated the location of the and expression of the A. sobria pap gene; and a comparison gene and its transcription direction. between this and the B. coagulans enzyme. Nucleotide Sequencing•\Sequence data were generated from single-stranded M13 or Bluescript subclones, contain ing inserts in both orientations, and deleted plasmids MATERIALS AND METHODS obtained by III digestion (22), by the dideox

Materials-Restriction enzymes, BAL 31 , T4 ynucleotide chain-termination method using Sequenase DNA , other modifying enzymes, kilo-sequencing (United States Biochemical). To overcome compression, dGTP was replaced by dITP, 7-deaza-dGTP (United States deletion kits, and primers for sequencing were from Takara Biochemical), or 7-deaza-dGTP and dATP mixes (Phar Shuzo, or Toyobo, and [35S]dCTP and [32P]dCTP were macia P-L Biochemicals) in some reaction mixtures. from Amersham. Sequenase was obtained from U.S. Routinely, 5% polyacrylamide gels containing 20% v/v Biochemicals, and Agarose I was from Dojin Chemicals. formamide were used for running sequencing reaction Proline ƒÀ-naphthylamide (Pro-ƒÀNA), hydroxyproline ƒÀ-naphthylamide (Hyp-ƒÀNA), Fast Garnet GBC salt, mixtures. Sequence data were analyzed using Genetyx Mac software (Software Development) and data base lysozyme, and RNAse A were from Sigma. Alkaline search was done using the "findget" and "iden" systems from calf intestine and Pseudomonas fragi from the National Institute of Genetics, Mishima. endoproteinase Asp-N were obtained from Boehringer Enzyme Activity Assay-The PAP activity was assayed - Mannheim. by measuring the amount of 8-naphthylamine liberated Bacterial Strains, Plasmids•\E. coli XL1-blue [supE44, from Pro-ƒÀNA, as described previously (23). One unit of hsdR17, recAl, endAl, gyrA46, thi, relAl, lac-, F•L(TnlO, the enzyme activity was defined as the amount of enzyme proAB, laclq ZM15)], DHl [F-, recAl, gyrA96, thi-1, releasing 1ƒÊmol of ƒÀ-naphthylamine per min. The protein hsdRl7 (rk-, mk-), supE44, relAl, 1-], and DH5a concentration was determined by the method of Bradford [supE44, ƒ¢lacU169 (ƒÓ580lacZƒ¢M15), hsdRl7, recAl, endAl, gyrA96, thi-1, relA1 ] were used as hosts. Plasmids (24) or by measuring the absorbance at 280 nm. For substrate specificity studies using peptide substrates, the pBR322, pFM005, pUC18, and pUC19 were used for liberated proline was determined using ninhydrin (25) or cloning and sequencing. Plasmid pFM005, a derivative of by HPLC. pKK223-3 (Pharmacia LKB Biotechnology) that bears the Culture of E. coli Transformants and Purification of strong tac promoter, was a generous gift of Dr. F. Misoka. Prolyl Aminopeptidase-E. coli harboring plasmid p14 or Phagemids Bluescript SK (+) and (-) (Stratagene) were used for routine cloning procedures, the construction of p15 was grown in 12 liters of N-broth [1% meat extract, 1% deletion mutants, and for nucleotide sequencing with polypeptone, and 0.5% NaCl, pH 7.0, containing ampicillin (50ƒÊg/ml)] in a fermentor at 30°C. Growth was followed single-stranded templates. Transformed E. coli was grown by measuring absorbance at 650nm. routinely at 30•Ž in Luria-Bertani broth (LB-broth) con All the purification procedures were done at 4°C. The taining ampicillin (50ƒÊg/ml). washed cells (50g wet weight) were suspended in 800ml of Construction of Genomic Library, and Screening-Aer 20mM Tris-HC1 buffer, pH 8.0 (Tris buffer), and disrupted omonas sobria was grown at 30•Ž in LB broth supplement for 10 min with glass beads in a Dyno-Mill. The glass beads ed with salts (0.05% magnesium sulfate, 0.001% ferrous were removed by decantation and the disrupted cell sus sulfate, 0.001% zinc sulfate, 0.0001% cupric sulfate, pension was centrifuged at 8,000•~g for 15min. The 0.0001% manganese sulfate, 0.0001% calcium chloride, and

Vol. 116, No. 4, 1994 820 A. Kitazono et al.

supernatant was treated with protamine sulfate solution constructed with inserts at the BamHI site of the vector (final concentration, 18 mg per gram of wet cells), then the showed high enzymatic activity. The insert in this clone was mixture was kept for 30 min and centrifuged. Ammonium found to be around 3kb long (pl); this fragment was sulfate was added to the supernatant to make it 40% isolated after BamHI and Smal double digestion and saturated, and after the solution had been allowed to stand ligated to similarly digested plasmids pUC18 and pUC19. for 1 h, the precipitated proteins were recovered by centri For the plasmids obtained (p4), the restriction enzyme map fugation. The pellet obtained was dissolved in Tris buffer was constructed (Fig. 1), and based on this, the fragment containing 20% saturation ammonium sulfate, and the clear containing the enzyme gene was further subcloned. The solution was applied to a column (6 x 15 cm) of Toyopearl gene was found to be transcribed in the BamHI•¨SmaI HW-65C equilibrated with the above buffer. The enzyme orientation, but the expression levels obtained were not as was not adsorbed, but passed through the column leaving a high as expected. Even after reduction of the length of the large fraction of the unwanted proteins on it. The active insert (p14), and subcloning into plasmid pFM005 harbor fractions were combined, and precipitated by adding ing the strong tac promoter, no significant increase in the ammonium sulfate to 80% saturation. The recovered enzymatic activity could be observed. To improve the precipitate was dissolved in Tris buffer; the resulting expression level, the construction of a gene fusion with the solution was desalted (Sephadex G-25) and applied to a ƒÀ-galactosidase N-terminal coded by the vector was at DEAE-Toyopearl column. Linear gradient elution was done tempted. Exonuclease III digestion was done from the with 0 to 0.5 M sodium chloride. BamHI site, and preparative agarose electrophoresis was Amino Acid Sequence and Composition of the Expressed run to separate the fragments of the desired length. After Enzyme-The amino-terminal region of the enzyme was ligation and transformation, the transformants were sequenced by Edman degradation (26, 27). Amino acid screened for PAP producers and among them, one showed sequences of some endoproteinase Asp-N peptides of the 18.4-fold higher enzymatic activity than the clone harbor expressed enzyme were also determined after isolation ing p4. In this plasmid (p15), the gene was in frame with the with an HPLC-Vydac C18 reversed phase column. Amino ƒÀ-galactosidase gene sequence and transcription occurred acid composition of the enzyme was determined after from the lac promoter on the vector, as was confirmed by hydrolysis with 6N HCl. nucleotide sequencing and amino acid sequencing of the Nucleotide Sequence Accession Number-The nucleotide expressed enzyme (not shown). This "fusion protein" was sequence data reported in this paper will appear in the GSDB, DDBJ, EMBL, and NCBI nucleotide sequence TABLE I. Taxonomic characterization of strain AK-5. databases with the accession number D30714.

RESULTS

Taxonomic Characterization of the PAP Producer Strain from Soil-Using Pro-ƒÀNA as the substrate, strains grown from soil samples were assayed for PAP activity. One strain was found to be a high producer of the enzyme, and was temporarily named AK-5. Table I shows the results of taxonomic analysis, which identified the strain as Aer omonas sobria, based on Bergey's manual (28). Isolation of the pap Gene, Subcloning•\Among 450 colonies of transformants analyzed, one from the library

Fig. 1. Restriction map of the

prolyl aminopeptidase gene from A. sobria. Based on this map a series of subclones was constructed, and the most representative are shown. The enzymatic activity of each is expressed as that relative to the clone bearing p4. In p15 the pap

gene is in frame with the ƒÀ-galacto sidase gene on the vector, producing high levels of the respective fusion protein.

J. Biochem. Prolyl Aminopeptidase Gene from Aeromonas sobria 821

Fig. 2. Primary structure of the prolyl aminopeptidase gene from A. sobria. The underlined regions are those found by amino acid sequencing of peptides obtained after endoproteinase Asp-N digestion of the purified recombinant enzyme. Upstream from the initiation codon, the putative promoter and ribosome-binding sites are also underlined. The N-terminal was found to be the first serine residue after the original methionine.

TABLE II. Purification of the A. sobria PAP from E. coli DH1/p15.

used to obtain peptides for partial amino acid sequence analyses. The "native" enzyme was prepared from the p14 clone and was used for characterization of the enzyme. The pap gene was found to occur as a unique gene, from the results of Southern hybridization analysis of the A. sobria genomic DNA (not shown). Nucleotide Sequence Determination and Analysis of A. sobria pap Gene-The complete nucleotide sequence of the gene was obtained for the insert in p14 (Fig. 2) by sequenc ing both strands of the DNA. An open reading frame of 1,275 nucleotides was identified from the sequence data. Fig. 3. Purification of the A. sobria prolyl aminopeptidase Seven bases upstream from the initiation codon ATG, a from E. coli DH1/p15. SDS-PAGE was run on 10% polyacrylamide sequence AGGAGA that could be acting as the ribosome gels, which were subsequently stained with Coomassie-Brilliant Blue binding site is present. Sequences that resemble the strong R250. Lane 1, protein size markers; lane 2, cell-free extract; lane 3, after 40% saturated ammonium sulfate precipitation; lane 4, pool promoters in E. coli could not be found, but the sequences after Toyopearl HW-65C; lane 5, pool after DEAE-Toyopearl elution. most likely to serve that function are underlined in the The protein size markers used were those of the "Low molecular figure. Deduced from this sequence is a protein having 425 weight calibration kit" from Pharmacia: phosphorylase b (94,000), amino acid residues and a calculated molecular mass of albumin (67,000), ovalbumin (43,000), carbonic anhydrase (30,000), 48,405 Da. All peptides sequenced from the purified en- trypsin inhibitor (20,100), and ƒ¿-lactalbumin (14,400).

Vol. 116, No. 4, 1994 822 A. Kitazono et al.

zyme were found in the deduced sequence (Fig. 2). Characterization of the Expressed Enzyme-The physi Moreover, amino acid composition analysis (not shown) cochemical properties of the purified Aeromonas native gave results that agreed with those predicted from the enzyme are shown in Table III, together with those for the nucleotide sequence. B. coagulans enzyme. The optimum pH and pH stability Purification of the Expressed Enzyme•\The purification values found were very close. However, the A. sobria procedure produced a preparation showing a single band on enzyme shows a very high optimum temperature and Coomassie Brilliant Blue R-250-stained SDS-PAGE (Fig. thermal stability (55 and 57•Ž, respectively). This deter 3). The results are shown in Table II for the purification mination was made using both the native and fusion enzyme from the clone harboring the p15 plasmid (fusion protein), with similar results. All these studies were conducted with with a recovery of 16%. The native enzyme could be Pro ƒÀNA as the substrate, under the conditions previously purified from the p14 clone with recoveries of less than 1% described (23). (not shown). For both enzymes, the amino-terminal se The isoelectric point shown in the figure was calculated quences were determined, confirming the presence of the from the amino acid sequence, and confirmed by isoelectric ƒÀ-galactosidase sequence for the fusion protein . The native focusing. A molecular weight of 205,000 was obtained by enzyme was found to begin with a serine residue, as was gel filtration (HiLoad 16/60 Superdex 200pg, FPLC expected considering the substrate specificity of the E. coli column), and one of 43,000 by SDS-PAGE, suggesting that methionine aminopeptidase. Amino acid sequence analysis the enzyme might be a homotetramer. of the peptides obtained by endoproteinase Asp-N digestion Effect of Chemicals on the Enzyme Activity and Sub gave results that were in agreement with those deduced strate Specificity-As is shown in Table IV, the Aeromonas from the nucleotide sequence (underlined regions in Fig. 2). enzyme was sensitive to PCMB, like the B. coagulans enzyme. The former was however, less affected by iodoace tic acid and manganese salts, and by denaturants such as TABLE III. Physicochemical properties of the prolyl amino urea and guanidine. Even at 5.4M urea concentration, 70% peptidases. of the activity remained after incubation. Other chemicals did not show any significant effect on the enzymatic activ-

TABLE V. Enzymatic activity on peptide substrates. N.A.: no activity was detected. APAP: Aeromonas sobria PAP; BCPAP: Bacillus coagulans PAP; PLSR12: PLSRTLSVAAKK; PPGF8: PPGFSPFR.

a70% of the enzymatic activity remained after incubation in each buffer and at room temperature for 30 min. b50% of the enzymatic activity remained after incubation at each temperature, and pH 8.0 for 15min.

TABLE IV. Effect of chemicals on the enzyme activity of A. sobria PAP. The enzyme was pre-incubated at 30°C for 10min with each additive, and the remaining activities were assayed under the standard conditions.

TABLE VI. Kinetic parameters of the expressed prolyl . N.A.. no activity was detected.

J. Biochem. Prolyl Aminopeptidase Gene from Aeromonas sobria 823

ity. The studies on substrate specificity were done in filtrates, suggesting the intracellular character of the parallel with the B. coagulans enzyme and the results are enzyme. The bacterium was found to be a high producer of shown in Tables V and VI. Both enzymes showed no activity the enzyme, and could be easily cultured when the medium on glycine, leucine, methionine, phenylalanine, pyro was supplemented with salts and the temperature of

glutamic acid, tryptophan, glycyl-proline, and alanine incubation was up to 30°C. The gene coding for PAP was f3NAs, poly-L-proline, or for protamines (salmine, Pro isolated and cloned in E. coli, and found to be composed of Arg,-Ser-Xaa22; clupeine, Pro-Arg4-Thr-Xaa25). However, 425 codons encoding a polypeptide of 48,405 Da. In an the enzymes showed different rates of hydrolysis of the attempt to improve the expression level and the production peptide substrates tested. For small peptides (up to three of the enzyme, a gene-fusion with the ƒÀ-galactosidase gene residues), the B. coagulans enzyme showed a higher fragment on the vector was constructed. As a result, a activity, but very low, or no proline liberation could be transformant overproducing the enzyme was obtained. detected for longer peptides (8 to 12 residues) (Table V). From the nucleotide sequence data, the pap gene was Likewise Hyp-ƒÀNA was not a substrate for this enzyme. lacking a promoter sequence resembling the E. coli consen The A. sobria enzyme, on the other hand, showed a higher sus sequence. Weak promoter activity could be hindering activity toward long peptides, and acted on Hyp-ƒÀNA with the overexpression of the gene. This problem was bypassed almost as high an activity as on Pro-ƒÀNA. only when a fusion plasmid was constructed, that is, when the gene was in frame with the ƒÀ-galactosidase enzyme, and transcription began from the lac promoter on the vector. DISCUSSION The expressed enzyme could then be purified in two forms: Aminopeptidases are present ubiquitously in cells and are in its native form with low yield, and as a fusion-protein essential for protein maturation and processing, degrada with a 16% recovery. Both purified forms showed a single tion of peptides, etc., and possibly determination of protein band of a relative molecular weight around 43,000 on stability. Present knowledge about aminopeptidases is SDS-PAGE. The value obtained by gel-filtration was about limited to the metalloenzymes, with leucine aminopep 205,000, suggesting that the enzyme occurs as a homo tidase (LAP) being the best studied and conserved group. tetramer. The enzyme had a high thermal stability and high LAP is a widely distributed zinc-dependent aminopep optimum temperature, differing in these aspects from the tidase and the first one for which a three-dimensional Bacillus, apricot seeds and Lactobacillus enzymes, which structure was determined (29); all its members are inhib are rather unstable. ited by bestatin. For bacteria and mammalians, most of the Another difference was found in their substrate speci aminopeptidases hitheeerto described belong to this group. ficities. The Aeromonas enzyme released not only proline Another important group is formed by the methionine but also hydroxyproline from the respective ƒÀ-naphthyl aminopeptidases, which are Cot+-dependent metallo-pep amides, and the amino-terminal proline from peptide tidases and are critical for the maturation of most proteins substrates of up to 12 residues. The activity towards longer in eukarvotic and prokaryotic cells. peptides could not be studied in detail, but no activity was On the other hand, few sulfhydryl aminopeptidases are detected towards poly-L-proline or for the protamines known, and among them, the only available amino acid (salmine, clupeine) tested. However, it was clear that the sequences are those for (dipeptidyl aminopep Bacillus, and the reported Lactobacillus and Neisseria tidase I) (30), pyroglutamyl aminopeptidase (31), and enzymes, were more active on short peptides (up to three residues), showing a decrease in their activities with an prolyl aminopeptidases (3, 4). Because of the scanty infor mation on SH-aminopeptidases, and taking into account the increase in the substrate length. Furthermore, the Bacillus and Lactobacillus enzymes were not active on Hyp-ƒÀNA specificity required for an enzyme to cleave peptide bonds involving proline residues, studies on prolyl aminopep and Hyp-Pro, respectively, presenting an absolute speci ficity for proline terminals. tidases are of great interest. In this report, we studied the From these results (Aeromonas and Bacillus enzymes) Aeromonas sobria PAP, cloning and sequencing the enzyme and those hitherto reported (Neisseria, Lactobacillus, and gene, and thereafter purifying and characterizing the apricot seed enzymes), the occurrence of at least two kinds expressed enzyme. The Aeromonas PAP activity was found to be unaffected of prolyl aminopeptidases can be proposed. One group might be formed by monomers of approximately 30kDa or by chelators, or by PMSF. It was strongly inhibited by rather small enzymes, comparatively unstable, being inhib PCMB and zinc chloride, results that were similar to those ited by some thiol-blocking reagent, and with a strict for the Bacillus (23) and Neisseria (32) enzymes, but it specificity for proline terminals. The second group consists showed less sensitivity to iodoacetic acid and manganese of stable, large enzymes of around 200kDa, with a broader chloride. DFP was to some extent inhibitory, but the specificity releasing also hydroxyproline terminals, a pre concentration required to inhibit the enzyme was more than ference for longer peptide substrates, and a variable ten times higher than that of PCMB (Table IV). It can be sensitivity to sulfhydryl enzyme inhibitors. To test this concluded that these three enzymes might have a cysteine hypothesis, we have purified and characterized the PAP residue in their . This finding agrees with those enzymes from three other sources: Hafnia aluei, Serratia reported for PAPs from other sources, i.e., apricot seeds marcescens, and Flavobacterium meningosepticum (data not (33), B. megaterium (34), bacteria from human oral cavity shown). The results confirmed that all these PAPs can be (35), and Propionibacterium acnes (36). All these studies classified in the two groups described above, on the basis of reported the inhibition of the enzymes by some thiol-block their molecular weight (size), and activity toward hydroxy ing reagent, and as a consequence, the PAP has been considered to be a sulfhydryl enzyme. proline terminals (substrate specificity stringency). The enzyme-genes have already been cloned and the determina- Aeromonas PAP activity was not detected in culture

Vol. 116, No. 4, 1994 _??_24 A. Kitazono et al.

Fig. 4. Alignment of the amino acid sequences of the A. sobria, cysteine (threonine for the Aeromonas enzyme), and the conserved B. coagulans, N. gonorrhoeae, and L. bulgaricus prolyl amino serine residue as indicated by Atlan et al. (5). APAP, BPAP, NPAP, _??_ eptidases. The amino acid residues that are conserved in the four and LPAP are respectively the Aeromonas, Bacillus, Neisseria, and enzymes are outlined. *, indicates the position of the conserved Lactobacillus prolyl aminopeptidase sequences.

_??_ion of the nucleotide sequences is in progress. the ester hydrolysis of atropine; and liver microsomal Before this report, there were three PAPs with known xenobiotic epoxide hydrolases from human and rat, which _??_rimary structures: the B. coagulans, the N. gonorrhoeae, inactivate metabolites of polycyclic aromatic hydrocarbons and the L. bulgaricus enzymes. The first two enzymes show generated by the cytochrome P450 system. The regions 23% identity, and the last two 29%; meanwhile, the that were conserved among these hydrolases were also Bacillus and the Lactobacillus enzymes have 36% identical observed in the PAPs aligned. Atlan et al. (5) explained residues. Although these values were reduced to values in this finding by postulating that the hydrolyses of cyclic t he range of 15-20% when the Aeromonas sequence was compounds and peptide bonds involving proline residues aligned, the conserved regions found for the first three might require a specific spatial arrangement of the region enzymes were remarkably well retained (Fig. 4). This surrounding the enzyme catalytic site. It would be of great _??_onservation was unexpected in view of the differences interest, then, to verify the presence or absence of these observed in size and in substrate specificity stringency hydrolase activities in the PAPs preparations, and vice _??_ nentioned above. The Bacillus and Neisseria enzymes are versa. both monomers of around 30kDa; the Lactobacillus was Atlan et al. (5) concluded the Lactobacillus enzyme to be reported to be a homotrimer with subunits of 34kDa; and a serine peptidase on the basis of the inhibition by he Aeromonas enzyme is formed by four subunits of 48 3,5-dichloroisocoumarin, as well as the conservation of the k Da each. sequence -GXSXGG observed in the alignment. On the The results from the inhibition studies prompted us to basis of these findings the Lactobacillus enzyme was ook for conservation of possible active residues. As a postulated to be a serine enzyme, and the inhibition by result, a threonine residue was found in the Aeromonas PCMB and heavy metals was explained as being caused by iequence at the position that was occupied by cysteine the alteration of conformational stability through the -esidues in the other three enzymes (number 62 for the oxidation of a thiol group directly or indirectly involved in Bacillus enzyme, Fig. 4). Whether this is the site of the the catalytic site. Reinforcing this argument was the fact fictive cysteine, and whether the threonine residue could act that the atropinesterase, whose amino acid sequence could is the nucleophile in the hydrolytic reaction by the Aer be aligned with those of the PAPs, had been shown to be a mtonas enzyme, remain to be determined. and its active serine (number 110) is It is also of great interest (5) that both the Lactobacillus conserved. The sequence is the consensus one found around nd Bacillus PAP amino acid sequences could be aligned the active serine for the members of the prolyl oligopep with a group of hydrolases showing different specificity: tidase family, and the same motif can also be found in the _??_-hydroxy-6-oxo-phenylhexa-2,4-dienoic acid hydrolase Aeromonas sequence. However, Gilbert et al. (6) did not _??_rom Pseudomonas putida, involved in polychlorinated detect any effect on the enzymatic activity of preincubating _??_iphenyl degradation; 2-hydroxymuconic semialdehyde the enzyme with DFP or PMSF, while the prolyl endopep _??_ ydrolase from Pseudomonas putida, which is involved in tidases and dipeptidyl peptidases IV members of the prolyl t he degradation of the dihydric phenol catechols resulting oligopeptidase family are all easily inhibited by DFP (18). from catabolism of aromatic compounds; atropinesterase Therefore, these data are not conclusive, and the studies from Pseudomonas putida, a serine hydrolase catalyzing made of the enzymes from other sources, seem to be in

J. Biochem. Prolyl Aminopeptidase Gene from Aeromonas sobria 825

agreement about the sulfhydryl character of the enzyme. Methods in Enzymology (Lorand, L., ed.) Vol. 45, pp. 404-415,

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