PEDS Advance Access published online on December 21, 2007
Protein Engineering Design and Selection, doi:10.1093/protein/gzm068
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Streptomyces aminopeptidase P: biochemical characterization and insight into the roles of its N-terminal domain
Research Institute for Biological Sciences (RIBS), Okayama, 7549-1 Kibichuo-cho, Kaga-gun, Okayama 716-1241, Japan
2 To whom correspondence should be addressed. E-mail: hatanaka{at}bio-ribs.com
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Abstract |
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We purified and characterized the aminopeptidase P from Streptomyces costaricanus TH-4 (thAPP). This enzyme has a tetramer structure, a metal-ion preference toward Zn, broad substrate specificity and a narrow pH dependency for activity. The primary structure of thAPP, respectively, exhibits 91% and 65% identity with those of two other APPs—APP I and APP II—from Streptomyces lividans (slAPP I and slAPP II). We next overexpressed the genes encoding thAPP and slAPP II in Escherichia coli and characterized them. Two differences were apparent in their properties: slAPP II formed a dimer, whereas thAPP formed a tetramer; also, the alkaline side pKa for the catalytic action of slAPP II is higher than that of thAPP. Investigation using chimeras of both enzymes revealed that the N-terminal domain is associated with the determination of pKa values for catalytic action and quaternary structure.
Keywords: aminopeptidase P/homology modeling/pKa value for catalytic action/quaternary structure/Streptomyces
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Introduction |
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Aminopeptidases form an abundant enzyme family in microorganisms (Gonzales and Robert-Baudouy, 1996
) and are associated with many biological functions such as protein degradation and protein maturation (Ben-Bassat et al., 1987; Chandu and Nandi, 2003). The expanded families of aminopeptidases, with distinct sequences and biochemical functions, match the chemical composition diversity of the substrate peptides (Rawlings et al., 2004, 2006). In the food industry, bacterial enzymes are valuable for the preparation of protein hydrolysates such as casein, collagen and gluten hydrolysates that are used as food ingredients and supplements. We have identified aminopeptidases from the genus Streptomyces (Arima et al., 2004; Hatanaka et al., 2007; Uraji et al., in press) and investigated their degradation of various peptides (Arima et al., 2006a). Broad-specificity aminopeptidases can release most N-terminal amino acids from peptides; however, they are unable to cleave imido bonds and consequently cannot release N-terminal amino acids when the penultimate residue is proline (Rul et al., 1994; McDonnell et al., 1999; Arima et al., 2006a). Consequently, they cannot degrade long peptides containing proline, which often imparts a bitter flavor to foods (Lemieux and Simard, 1991). Proline-specific aminopeptidases are therefore required to remove the blockage to the further action of aminopeptidases as presented by proline residues in the second position of the N-terminus in peptides. For that reason, we specifically examined aminopeptidase P (APP), which is a metalloprotease that cleaves the N-terminal amino acid residue from a peptide in which the penultimate residue is proline.
As a model to clarify the mechanism of action of this class of enzymes, APP was studied because APPs play an important role in the regulation of biologically active peptides that have Xaa–Pro at the N-terminus such as the potent vasodilatory peptide hormone bradykinin. The crystal structure of the APP from Escherichia coli has already been characterized at a high resolution (Wilce et al., 1998; Graham et al., 2003); furthermore, its catalytic mechanism has already been studied extensively (Graham et al., 2005, 2006; Jao et al., 2006). In addition to its previously described biomedical importance, obtaining further information on such enzymes, including the detailed functions of their regions and residues, is useful to develop potent biocatalysts through modification of their properties using protein engineering techniques. Therefore, further information related to homologous enzymes is of interest in the industrial field.
Our recent study demonstrated that Streptomyces costaricanus TH-4 secretes numerous extracellular proteins (Hatanaka et al., 2007); we also found APP (thAPP) activity in the culture supernatant. In this study, we cloned and expressed the gene encoding thAPP. The primary structure of thAPP, respectively, exhibits 91% and 65% identities with those of two other APPs: APP I and APP II from Streptomyces lividans (slAPP I and slAPP II). Although the cloning of the genes encoding these two APPs has already been reported (Butler et al., 1993, 1994), their properties remain unclarified. Therefore, we characterized thAPP and slAPP II to obtain detailed information related to their biochemical properties. Furthermore, we constructed chimeras of the two APPs to analyze the relationship between the differences in their properties and domains.
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Materials, bacterial strains and plasmids
A snake venom L-amino acid oxidase (LAAO) was purchased from Sigma Chemical Co. Horseradish peroxidase was purchased from Wako Pure Chemical Industries Ltd. Peptides and dipeptidyl p-nitroanilides (pNAs) were obtained from Bachem A.G. Recombinant proline aminopeptidase (PAP) from Streptomyces aureofaciens TH-3 (TH–PAP) was obtained as follows: E. coli BL21(DE3) harboring the expression vector for PAP production, i.e. pET–THPAP (Uraji et al., in press), was cultivated at 25°C for 24 h in 50 ml of Overnight ExpressionTM Instant TB medium (Novagen Inc.). Then TH–PAP was purified from the harvested cells according to the procedures described by Uraji et al. (in press).
As the donor strain for thapp, S. costaricanus TH-4 was used. The plasmid pCR–Blunt II–TOPO (Invitrogen Corp.) was used as the cloning vector. The plasmids pET-22b and pET-24b (Novagen Inc.) were used as expression vectors. As the host strain for general cloning procedures, E. coli JM109 was used; E. coli BL21(DE3) was used as the host strain for gene expression.
The APP activity toward Xaa–Pro-containing peptides was determined through quantitative assay of amino acids derived from the hydrolyzed substrates. The constant for determination of the amino acid concentration, which was obtained from the standard curve for amino acids, was referred from our previous report (Arima et al., 2006a). An enzyme solution (0.1 ml) and a substrate solution (0.1 ml, 50 mM) were added to 0.8 ml of 100 mM Tris–maleic acid (pH 7.4). The reaction mixture was incubated at 30°C for 5 min, then the reaction was stopped using heat treatment (95°C, 5 min). The liberated amino acid Xaa was detected using the 4-aminoantipyrine phenol method (Allain et al., 1974), coupled with the reaction of L-amino acid oxidase. After centrifugation for 5 min, 100 µl of the supernatant was added to 0.9 ml of a mixture containing 100 mM Tris–HCl (pH 8.0), 0.5 mM 4-aminoantipyrine, 1.7 mM phenol, 50 µg/ml horseradish peroxidase and 0.2 mg/ml snake venom L-amino acid oxidase, which can oxidize widely various hydrophobic amino acids (Massay and Curti, 1967). After incubation for 2 h at 37°C, the absorbance of the solution at 505 nm was determined. The initial reaction rate (v, in µmol min–1 mg–1) of APPs was calculated from the observed absorbance at 505 nm (A, in AU), the enzyme concentration in the enzyme solution (c, in mg/ml), reaction time (t, in min) and the constant 6.096, obtained from the standard curve for amino acids, according to the formula v = 100A/6.096ct.
The activity of APP toward Xaa–Pro–pNA was determined under the following conditions. An enzyme solution (0.1 ml) and a substrate solution (0.1 ml, 50 mM) were added to 0.8 ml of 100 mM Tris–maleic acid (pH 7.4) containing TH–PAP (50 µg/ml). The mixture was incubated at 30°C. The liberated Pro–pNA was detected simultaneously using the TH–PAP reaction, which can hydrolyze Pro–pNA efficiently (Uraji et al., in press). The increase in absorption at 405 nm caused by the release of p-nitroaniline per minute was monitored continuously using a spectrophotometer (U2800; Hitachi Ltd). The initial activity rate was determined from the linear part of the optical density profile (
405 nm = 10 600 M–1 cm–1 (Prescott and Wilkes, 1976).
Purification of thAPP from culture supernatant of S. costaricanus TH-4
In a tryptic soy broth, S. costaricanus TH-4 was grown aerobically at 30°C for 72 h. The culture supernatant was brought to 70% saturation with ammonium sulfate; the resultant precipitate was then dissolved in 20 mM Tris–HCl (pH 8.0) containing 25% ammonium sulfate. This solution was loaded onto a Butyl Sepharose 4 Fast Flow column (Amersham Pharmacia Co. Ltd. Biotech) that had been equilibrated with 20 mM Tris–HCl (pH 8.0) containing 25% ammonium sulfate. The column was washed with the equilibration buffer and then eluted with 10% ammonium sulfate. Fractions exhibiting high activity were pooled, and then dialyzed against 20 mM Tris–HCl (pH 8.0). The dialyzed solution was loaded onto a DEAE Sepharose Fast Flow column (Amersham Pharmacia Co. Ltd. Biotech) equilibrated with 20 mM Tris–HCl (pH 8.0). The column was washed with 200 mM NaCl, then the enzyme was eluted using 350 mM NaCl. Fractions exhibiting high activity were pooled, and then dialyzed against 20 mM Tris–HCl (pH 8.0). Next, the dialysate was loaded onto a Vivapure-Q spin column (Millipore Corp.) equilibrated with 20 mM Tris–HCl (pH 8.0). After washing with 200 mM NaCl, the bound protein was eluted with 400 mM NaCl. The eluent was then loaded onto a gel-filtration column (Superdex200; Amersham Pharmacia Co. Ltd. Biotech). The APP activity was monitored in terms of Met–Pro hydrolytic activity. The APP purified using these procedures was used for sequence analysis and characterization.
Determination of N-terminal amino acid sequences
The purified thAPP was blotted onto a PVDF membrane after 10% SDS–PAGE under denaturing conditions. The membrane was then stained using Coomassie brilliant blue; the protein band was excised from the membrane. The protein band was sent to APRO Science, from whom we requested the determination of the N-terminal sequence by Edman degradation.
We aligned the sequences of the known APPs from Actinomycetes using the CLUSTAL algorithm to design the primer. To obtain the gene thapp, the forward primer [5'-GACGAGCT(CG)AACCC(CG)GAGAC(CG)CC(CG)GA-3'] was designed using the N-terminal amino acid sequence of thAPP (-SDELNPETPE-); the reverse primer [5'-GTCCTCGAT(CG)CG(CG)AC(CG)CCGAT-3'] was designed using the sequence of the homology domain of APPs from Actinomycetes (-IGVRIED-). After PCR amplification using chromosomal DNA, we determined a partial sequence of thapp using the amplified DNA fragment. Next, a digoxigenin-labeled PCR probe was synthesized using a synthesis kit (PCR DIG Probe; Roche Molecular Biochemicals). In addition, DNA hybridization on a membrane was performed. First, 2.2 kb fragments digested with SphI were hybridized with the probe. The fragments were recovered and self-ligated. Then the ligation products were amplified using PCR with the primer set (5'-ACCGAGACGTACGTGGAGGGTGTGCTGGAG-3' and 5'-CTGCATGTTCTCGGCCAGCTCGTCGGAGAC-3') designed from the partial sequence of thapp. The PCR product was then used for sequencing. The entire sequence of thapp has been assigned the Accession No. AB284164 [GenBank] in the DDBJ database.
Construction of expression vectors
The full-sized genes encoding thAPP and slAPP II were amplified using PCR from the chromosomal DNAs of S. costaricanus TH-4 and S. lividans, respectively, using the following primers: 5'-CATATGTCCGACGAGCTGAACCCGGAGACC-3' and 5'-GGATCCTCAGCCCTTCAGCGAGGCCATCCA-3' for the amplification of thapp, and 5'-CATATGTCGAACCGTCGGAAGAACAGCCTG-3' and 5'-GGATCCTCAGCCCGCGAAGCGGGCCATCCA-3' for amplification of the gene encoding slAPP II (the underlined regions are NdeI or BamHI sites). The PCR products were cloned into pCR-Blunt II-TOPO and the correct cloning was confirmed using sequencing. Both genes were then subcloned into the NdeI–BamHI gap of pET-22b, respectively, yielding pET–thAPP and pET–slAPPII.
Gene expression of recombinant APPs
We cultivated E. coli BL21(DE3), harboring the constructed expression vector for APP production, at 22°C for 24 h in 100 ml of Overnight ExpressionTM Instant TB medium (Novagen Inc.), a medium designed for an IPTG-inducible bacterial expression system which obviates monitoring of cell growth and addition of IPTG. The harvested cells were suspended in 20 mM Tris–HCl (pH 8.0), and then disrupted by ultrasonication on ice. After removal of cell debris, APP was purified according to the procedures described in the subsection ‘Purification of thAPP from culture supernatant of S. costaricanus TH-4’.
Two halves of the 5'-end domain and 3'-end domain of slappII were amplified using the following primers: 5'-CATATGTCGAACCGTCGGAAGAACAGCCTG-3' and 5'-GAGCTCCCCGAGTTCCCAGGCGTCCTTGAC-3' for amplification of half of the 5'-end domain, and 5'-GGATCCTCAGCCCGCGAAGCGGGCCATCCA-3' and 5'-GAGCTCCGCAAGGCGGTGGACTCCACCGTG-3' for amplification of half of the 3'-end domain (the underlined regions are NdeI, SacI and BamHI sites). The PCR products were cloned into pCR-Blunt II-TOPO, and the insert fragment was separated from each obtained plasmid. Next, using NdeI–SacI, or SacI–BamHI digestion of pET–thAPP, two halves of the 5'-end and 3'-end domains of thapp were prepared. The fragments of half of the 5'-end domain of thapp and half of the 3'-end domain of slappII were ligated into the NdeI–BamHI gap of pET-24b. Similarly, the halves of the 5'-end and 3'-end domains of slappII and thapp, respectively, were ligated into the NdeI–BamHI gap of pET-24b [yielding pET-(th–sl)cAPP and pET-(sl–th)cAPP, respectively].
The molecular weight of thAPP was determined using SDS–PAGE and gel-filtration analyses. Gel filtration was performed using a Superdex 200 10/300 GL column (Amersham Pharmacia Co. Ltd. Biotech) equilibrated with 20 mM Tris–HCl (pH 8.0) containing 0.2 M NaCl. The proteins used as standards of the molecular weight were glutamate dehydrogenase (290 kDa), lactate dehydrogenase (142 kDa), enolase (67 kDa), myokinase (32 kDa) and cytochrome C (12.4 kDa). The proteins were eluted at a flow rate of 0.5 ml/min. The effect of pH on enzyme activity was examined at 30°C in 80 mM Tris–malate (pH 5–8.6) and Gly–NaOH (pH 8.6–10.6). Thermostability was tested by incubating 100 µl of a sample (10 µg/ml protein) in 20 mM Tris–HCl (pH 8.0) for 30 min at temperatures of 16–50°C. Residual activity was measured using Ala–Pro–pNA under the conditions described in the ‘Enzyme assay’ subsection. Kinetic parameters were determined in a mixture containing 0.1 ml of an enzyme solution (50 µg/ml), 0.1 ml of a 2–64 mM Ala–Pro–pNA solution and 0.8 ml of 100 mM Tris–maleic acid (pH 7.4) containing 10 U/ml THPAP under the conditions described in the ‘Enzyme assay’ subsection. By treating the enzyme with 50 mM EDTA overnight at 4°C, apo-formed thAPP was prepared and the treated samples were dialyzed against 10 mM Tris–HCl (pH 8.0). The effect of metal ions on the activity of the apo enzyme was determined by adding a metal ion (Zn, Co or Mn) at an appropriate concentration, incubating the mixture overnight at room temperature, and performing an assay to determine the APP activity of the treated samples.
The structures of the Streptomyces APPs were built using a homology modeling method of Internal Coordinate Mechanics (ICM) software (MOLSOFT), which is a program for comparative protein structure modeling and interactive docking. For modeling of the structure of Streptomyces APPs, structural data of the APP from E. coli (PDB ID, 1M35 and 2BHD) were used as a template.
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Purification and characterization of thAPP
We purified thAPP from the culture supernatant of S. costaricanus TH-4 to obtain information related to its characteristics. As presented in Table I, four runs of column chromatography (Butyl-Sepharose, DEAE-Sepharose, Vivapure-Q and gel filtration) were performed, and thAPP was purified to homogeneity, as determined by SDS–PAGE (Fig. 1A). thAPP showed molecular masses of 
55 kDa on SDS–PAGE and 220 kDa on gel filtration (Fig. 1A and B), indicating that thAPP is a tetramer.
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Figure 2A and B shows that thAPP is stable at pH 5.0–9.0; it exhibited moderate stability with respect to temperature (up to 32°C). Its activity was optimum at pH 6.5–8.0 (Fig. 2A) and at temperatures of 30–40°C. In an investigation using various Xaa–Pro dipeptides, Xaa–Pro–Yaa tripeptides and Xaa–Pro–pNA derivatives, thAPP was found to have broad specificity (Fig. 2C). Among them, th-APP showed the highest activity toward Ala–Pro–pNA. The results of kinetics analyses performed under the optimal pH (7.4) at 30°C are summarized in Table II.
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The activity of thAPP was inhibited by the addition of EDTA (Fig. 2D), indicating that thAPP formed an apo enzyme as a result of the treatment. Figure 2D shows that the activity of the apo-formed thAPP was recovered by
90% by adding 100 µM Zn. However, the addition of more Zn inhibits the activity. The activation was also observed with the addition of Co or Mn, but the activity did not fully recover. The N-terminal amino acid sequence of thAPP was determined to be SDELNPETPE. Aligning the sequence of thAPP with those of the known APPs from Actinomycetes indicated that a homologous sequence exists, i.e. RGIGVRIED, among these APPs in their C-terminal region. Using this sequence information, we designed PCR primers and analyzed the sequence of the gene encoding thAPP using an inverse PCR technique. In the determined nucleotide sequence, the initiation codon, GTG, was found neighboring the sequence which codes the determined N-terminal sequence of thAPP (data not shown), thereby indicating that no signal peptide for secretion (Fig. 3). The secretion of thAPP was presumably caused by mycelial lysis. The deduced amino acid sequence of thAPP, respectively, showed 91%, 66% and 33% identities with those of two APPs from S. lividans (i.e. slAPP I and slAPP II) and the APP from E. coli (Fig. 3). Five amino acid residues observed in the peptidase M24 family, which are involved in the coordination of the two metal atoms, were found in thAPP and in other Streptomyces APPs.
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Overproduction of thAPP and slAPP II
Because slAPP I shows 91% identity with thAPP, slAPP I is considered to have the same characteristics as thAPP. To obtain basic molecular information related to Streptomyces APPs, we chose slAPP II, the primary structure of which has 66% identity with thAPP, as an enzyme for comparative analysis of Streptomyces APPs. We constructed two expression plasmids (i.e. pET–thAPP and pET–slAPPII) to overexpress thapp and slappII in E. coli. Although SDS–PAGE analysis of the cell lysate indicated that the E. coli transformant expressed a large amount of the appropriate protein, no (or significantly low) APP activity exists in the cell lysate. The APP activity was detected by the addition of Zn. For that reason, we considered that the recombinant APPs produced by E. coli formed an apo enzyme. Therefore, treatment with 100 µM Zn was needed to recover APP activity before purification. The recombinant enzymes were expressed up to a concentration equivalent to 30% of the total soluble cellular protein concentration (data not shown). The kcat of the purified recombinant thAPP was one-fifth lower than that of the thAPP purified from S. costaricanus TH-4 (Table II). This difference is considered to result from inactivation of the enzyme, which lacks metal ions in its active site, during the E. coli transformant cultivation. Similar to that in thAPP, the activation induced by Zn addition was observed in the recombinant slAPP II; the addition of Co and Mn led to only a slight activation (data not shown).
Comparative analysis of recombinant thAPP and slAPP II
We next characterized both recombinant APPs and compared their properties. The properties of recombinant thAPP, such as its respective stabilities toward pH and temperature, effects of metal ions on activity and the substrate specificity, were similar to those of wild-type thAPP (data not shown). Recombinant slAPP II is stable at pH 5.0–9.0, and exhibited moderate stability with respect to temperature (up to 30°C), the data were similar to those of thAPP (data not shown). In an investigation of substrate specificity, slAPP II shows strict specificity toward Met–Pro among various Xaa–Pro dipeptides. In contrast, an investigation using Xaa–Pro–Yaa tripeptides and Xaa–Pro–pNA dervatives indicates a broad specificity (Fig. 4). Consequently, data related to the substrate specificity of slAPP II were similar to those of thAPP.
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In contrast to the characteristics of slAPP II described above, slAPP II showed an 8.7-fold lower hydrolysis rate toward Ala–Pro–pNA than thAPP (Table II). In addition to the reaction rate difference, we found two differences in their properties. First, slAPP II showed molecular masses of
52 kDa on SDS–PAGE and 110 kDa on gel filtration (Fig. 5A and B), indicating that slAPP II is a dimer, whereas th-APP is a tetramer. Secondly, the effect of pH on the slAPP II activity differed from that in the case of thAPP (Fig. 6A and B). That is, the pH dependency of slAPP II for Ala–Pro–pNA hydrolysis was wider on the alkaline side than that of thAPP. We determined pKa values by plotting logV versus pH (Dixon plots), which indicated that the pKa values of ionizable residues associated with catalytic action in both enzymes (on the alkaline side, the pKa values for catalytic action were 8.0 ± 0.1 and 8.8 ± 0.1, respectively, for thAPP and slAPP II) (Fig. 6A and B). We also investigated the effect of pH on Km for Ala–Pro–pNA hydrolysis. Both enzymes showed similar pH dependence of Km (Fig. 6A and B). For that reason, the difference in the pH dependency for Ala–Pro–pNA hydrolysis is considered to be independent of the pKa values of ionizable residues associated with substrate binding.
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Construction of chimeric APPs
The tertiary and quaternary structure of the APP from E. coli has already been clarified (Wilce et al., 1998; Graham et al., 2003); this enzyme has a tetramer consisting of a dimer of dimers. The APP protomer structure is divided between the C-terminal domain containing the catalytic site and the N-terminal domain. A part of the N-terminal domain is responsible for connections between subunits (Wilce et al., 1998) (Fig. 7A). Therefore, the N-terminal domain of APP was responsible for the function of determining the quaternary structure. In fact, we used the homology modeling capability of ICM software with the structure of the APP from E. coli as a template to construct the Streptomyces APP structures. In the structures, thAPP shows that the dimer–dimer attachment site of the N-terminal domain appears to be well fitted. However, the predicted structure of slAPP II shows a crevice at the dimer–dimer attachment site (data not shown). In terms of determining the pKa values for catalytic action, the residues existing in the C-terminal domain were inferred to affect the shift of pKa because the catalytic sites are composed mainly of the C-terminal residues. However, in the structure of the APP from E. coli, two chains in the N-terminal domain extend to the catalytic sites of another subunit (Fig. 7B). At this point, we could not predict which domain is associated with the difference in the pKa values.
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To verify which domain is associated with the differences in the properties of both Streptomyces enzymes, we constructed two chimeric APPs: th–sl cAPP, in which half of the N-terminal domain is thAPP and the rest is slAPP II and sl–th cAPP, in which half of the N-terminal domain is slAPP II and the rest is thAPP. The amino-acid-based chimera-forming site is presented in Fig. 3. The two chimeric APPs were produced using E. coli as an active form; both chimeric APPs also showed the activation induced by Zn addition. Using the same procedures used for thAPP purification, both chimeric APPs were purified to homogeneity (Fig. 5A).
Comparative analysis of chimeric APPs
Using a kinetic analysis of the chimeric APPs, extremely low activities were observed at the optimal pH (Table II). Consequently, we were unable to identify the domain associated with the difference in the reaction rate. For this investigation, we examined the quaternary structures and the pH dependency of the activities of both chimeric APPs.
Gel-filtration analysis results showed that th–sl cAPP and sl–th cAPP, respectively, form a tetramer and a dimer (Fig. 5C), which indicates that the N-terminal domain is associated with the determination of quaternary structure. We next examined the pH dependence of the activities and observed bell-shaped curves. As presented in the upper panels of Fig. 6C and D, the pH dependence and pKa values of th–sl cAPP were almost identical to those of thAPP. In contrast, sl–th cAPP showed curves and pKa values that resembled those of slAPP II. Similar to those of the wild-type enzymes, the Km values of both chimeric APPs showed almost equivalent pH dependence (lower panels of Fig. 6C and D). These results suggest that the N-terminal domain is also associated with the determination of the pKa values for catalytic action.
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Discussion |
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In this study, we first characterized thAPP purified from the culture supernatant of S. costaricanus TH-4. Although cloning of the genes encoding two APPs from S. lividans has already been reported (Butler et al., 1993
, 1994), this study yielded novel findings related to the basic enzymological characteristics of APPs such as quaternary structure, pH stability, thermal stability, substrate specificity and the preference of APPs toward metal ions. They showed similar results to those of the APP from E. coli, i.e. having a tetramer structure and broad substrate specificity. However, the thAPP preference toward metal ions differs from that of the APP from E. coli. The APP from E. coli has been well studied as an authentic enzyme of the peptidase M24 family. Of the bacterial enzymes of the peptidase M24 family, the methionine aminopeptidase from E. coli is also well characterized. Both Mn and Co are present in the catalytic center in the APP and methionine aminopeptidase from E. coli, respectively (D’souza et al., 2000; Cosper et al., 2001; Larrabee et al., 2004; Graham et al., 2005). Streptomyces APPs showed the five conserved amino acid residues, which are observed in the peptidase M24 family, involved in the coordination of two metal atoms (denoted with an asterisk in Fig. 3). In contrast, an investigation of the preference toward metal ions revealed that the activity of the apo-formed thAPP was recovered fully by adding 100 µM Zn. However, adding more Zn led to the inactivation of the enzyme. In addition, the presence of other metal ions did not fully recover the activity of the apo-formed thAPP. Identical results were obtained for thAPP and slAPP II (data not shown). Therefore, unlike other bacterial enzymes belonging to the peptidase M24 family, we consider that Streptomyces APP originally had Zn ions in its active site. By characterizing two APPs from Streptomyces, two differences in their properties besides the difference in their reaction rates were found: differences in their pKa values for catalytic action and quaternary structures. The pH dependence of the activity of slAPP II was wider on the alkaline side than that of thAPP (Fig. 6A and B), and thAPP and slAPP II were found, respectively, to have tetramer and dimer structures. In addition, an investigation using chimeric APPs showed that the N-terminal domain of both Streptomyces APPs is associated with the determination of pKa values for catalytic action and quaternary structure.
In the structure of APP from E. coli, part of the N-terminal domain is responsible for connections between subunits (dimer of dimers) (Wilce et al., 1998). Entangled chains are observed at these dimer–dimer attachment sites of the APP from E. coli. The enzyme is considered to maintain its tetramer structure tightened by these interactions in the N-terminal region (Fig. 7A). The results of the homology modeling exhibit that, in thAPP, the dimer–dimer attachment site of the N-terminal domain appears to be well fitted. In contrast, slAPP II shows a crevice at the dimer–dimer attachment site (data not shown), which indicates that the dimer–dimer attachment site of slAPP II is not fitted well. Because of this structural difference, we consider that slAPP II loses the contact function of the N-terminal domain, engendering dimer formation.
The substrate-binding pocket of APP includes residues in the C-terminal domain (Wilce et al., 1998; Graham et al., 2004). In these residues, His243, His350, His361 and Arg404 are considered to be candidates for determining pKa values for catalytic action. That is, they are associated with the interaction with substrate (His243 and His361), modulation of the Lewis acidity of the active site metal atoms (His361) or determining the electrostatic environment around the penultimate proline of substrate (His350 and Arg404). However, the investigation using chimeric APPs demonstrated that the pKa values for catalytic action were changed by the interchange of the N-terminal domain. Therefore, the residues of Streptomyces APPs corresponding to the four candidate residues of E. coli described above are not associated with the shift of the pKa value. In the E. coli APP structure, two chains existing in the N-terminal domain (the regions of Pro31–Tyr43 and Arg79–Leu93) are extended to the catalytic site of another subunit (Fig. 7B). Graham et al. (2006) described in a previous report that the side chain of Asp38 of E. coli APP, which is included in one of the regions, Pro31–Tyr43, fixes the His361 imidazole ring of the adjacent subunit. Because His361 is considered to have a role in optimally positioning the scissile peptide bond for nucleophilic attack, the role of Asp68 is also considered important in the catalytic action. However, the residue corresponding to Asp38 of E. coli is conserved between thAPP and slAPP II (Fig. 3). Consequently, we concluded that this residue is also not associated with the pKa value shift.
Alignment analysis of Streptomyces and E. coli APPs raises the possibility that the structural differences in the two chains existing in the N-terminal domain of both Streptomyces APPs, which are extended to the catalytic site of another subunit, are associated with the shift of the pKa value for catalytic action. In thAPP, Asn80–Phe92 and Arg131–His150 correspond, respectively, to Pro31–Tyr43 and Arg79–Leu93 of E. coli APP. In slAPP II, Asn64–Phe76 and Arg116–Arg135, respectively, correspond to the former and the latter (Fig. 3). Between thAPP and slAPP II, the residues of the former regions are conserved more than those of the latter region. In particular, N-terminal 12 residues of the latter region indicate eight residues differences. Moreover, comparison of the structures of Streptomyces APPs constructed using homology modeling showed that the catalytic site of thAPP is narrower than that of slAPP II by the bulkiness of the chains from the N-terminal of another subunit (data not shown). We speculate that these structural differences cause the differences in pKa values for catalytic action.
Besides the domains extended to the catalytic site of another subunit, both Streptomyces APPs show appreciably different N-terminal parts. In particular, thAPP has additional region of 16 residues at N-terminal end that were undetected in the slAPP sequence (Fig. 3). In addition, the recombinant protein contains in the first position either formyl methionine or methionine, or no initiation methionine at all. The situation involving initial methionine can be different for different proteins, which will lead to additional differences in N-terminal domains. Because N-terminal end is distant from the catalytic site and the position of the dimer–dimer interactions, we consider the difference in N-terminal end cannot be associated with the determination of pKa and quaternary structure. However, the characteristics of Streptomyces APPs were similar but not the same. We consider the difference of the sequence in N-terminal domain is associated the subtle difference in the characteristics of both Streptomyces APPs.
This study demonstrated an extremely important difference between the two Streptomyces APPs: their reaction rates. In fact, thAPP showed a reaction rate for Ala–Pro–pNA hydrolysis that was 8.7-fold higher than that of slAPP II (Table II). Unfortunately, because the chimeric APPs activities were extremely low, we were unable to identify the domain associated with the difference in the reaction rate in this study. We speculate that subtle differences in the electrostatic and steric environments and in the distances between the atoms forming the ionic network around the catalytic center of each enzyme are the cause of that 8.7-fold difference in reaction rates. That is, the fine tuning of the conformation of the catalytic site and the interaction between the enzyme and the substrate are probably important for determining the reaction rate. This speculation is in agreement with the results of the chimeric APPs, which showed extremely low activities.
In this study, we obtained two APPs with broad substrate specificities from Streptomyces. Bacterial APPs are useful for preparation of protein hydrolysates, i.e. removing the blockage to the further action of broad-specificity aminopeptidases as presented by proline residues in the second position of the N-terminus in peptides. Although these APPs show broad specificity, they are only useful as biocatalyst in a limited pH range. The pH of the enzyme reaction is important when using enzymes as biocatalysts in the industrial field. For that reason, the characteristics of APPs from Streptomyces, in terms of the effect of pH on activity, should be improved for industrial use. In our recent studies, using chimeragenesis and site-directed mutagenesis, we carried out the modification of enzyme functions to tailor an enzyme to become a potent biocatalyst (Arima et al., 2005, 2006b, 2006c, 2006d). Further study to identify important residues related to pKa determination is necessary to develop APPs from Streptomyces as excellent biocatalysts.
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TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingReferences |
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Research fund from Okayama Prefecturol Government.
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1 Present address: Department of Agricultural Chemistry, Faculty of Agriculture, Tottori University, Tottori 680-8553, Japan
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Received June 12, 2007; revised October 30, 2007; accepted October 31, 2007.
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