Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Supplemental material Supplemental material Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Chao, S.-H. Articles by Tsai, Y.-C. Search for Related Content PubMed PubMed Citation Articles by Chao, S.-H. Articles by Tsai, Y.-C. Agricola Articles by Chao, S.-H. Articles by Tsai, Y.-C.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, January 2006, p. 968-971, Vol. 72, No. 1
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.1.968-971.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Characterization of a Novel PepF-Like Oligopeptidase Secreted by Bacillus amyloliquefaciens 23-7A

Shiou-Huei Chao,¹ Tzu-Hao Cheng,¹ Chin-Ying Shaw,¹ Meng-Hwan Lee,¹ Yuan-Hsun Hsu,² and Ying-Chieh Tsai^1*

Institute of Biochemistry, National Yang-Ming University,¹ Graduate Institute of Medical Science, Taipei Medical University, Taipei, Taiwan²

Received 19 April 2005/ Accepted 6 October 2005

Top Abstract Introduction References

 
An oligopeptidase from Bacillus amyloliquefaciens 23-7A was characterized along with its biochemical activities and structural gene. The protein's amino acid sequence and enzymatic activities were similar to those of other bacterial PepFs, which belong to metallopeptidase family M3. While most bacterial PepFs are cytoplasmic endopeptidases, the identified PepF_Ba oligopeptidase is a secreted protein and may facilitate the process of sporulation.

Top Abstract Introduction References

 
In various species and tissues, peptidases of family M3 are involved in peptide degradation, bioactive neural-peptide synthesis, and cleavage of signal peptides (3, 4, 14, 16). This kind of endopeptidase only hydrolyzes oligopeptides that contain no more than 20 amino acid residues. Bacterial PepFs also belong to the M3 family of peptidases. In this report, another PepF-like oligopeptidase was identified in a collagen-degrading strain, Bacillus amyloliquefaciens 23-7A. This peptidase, designated as PepF_Ba, was characterized in great detail, and its potential physiological roles were also discussed.

Bacterial cultivation and enzyme purification.
The microbial strain 23-7A bearing collagenolytic activity was screened from soil in Taiwan. The bacterium was spore-forming, gram-positive, and taxonomically identified as Bacillus amyloliquefaciens by 16S rRNA gene sequence and API strips. During cultivation of this strain in the basal medium (1% defatted soybean as the nitrogen source, 1% glucose, 0.5% yeast extract, 0.1% K₂HPO₄, and 0.2% MgSO₄) at 37°C using a Biostat B 5-liter fermentor (Sartorius BBI Systems Inc.), the cell density and number of spores were determined. Since PepF_Ba is the only secreted protease that is able to cleave synthetic substrate N-(3-[2-furyl]acryloyl)-Leu-Gly-Pro-Ala (FALGPA) in this strain (data not shown), proteolysis of FALGPA was used to monitor PepF_Ba that was present in the supernatant of collected cell pellets. PepF_Ba activity appeared at the beginning of exponential phase and lasted until spores began to form (Fig. 1). Purification of this enzyme from the cell supernatant was carried out with a cultivation time of 16 h and subjected to a series of procedures (see the supplementary material). PepF_Ba was purified 1,035-fold with a total recovery of 4.6% (see Table S1 in the supplementary material). The purified protein has a molecular mass of 69,000 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the native molecular mass was estimated to be 170 kDa by gel permeation chromatography on a Superdex 200 HR column, suggesting that PepF_Ba forms a homodimeric molecule (see Fig. S1 in the supplemental material).

View larger version (14K): [in this window] [in a new window]

FIG. 1. PepFBa activity and optical density of B. amyloliquefaciens 23-7A grown in basal medium. Total cell numbers () at the indicated time were measured by optical density at 600 nm (OD600). For spore titer () determination, the cultured broth was heated for 20 min at 65°C and then spread at appropriate dilutions on LB agar plates. PepFBa activity () was determined by hydrolysis of the peptide FALGPA as described previously (17).

Amino acid sequence, molecular cloning, and DNA sequencing.
The N-terminal sequence of purified PepF_Ba was determined by automatic sequencing to be Ser-Glu-Lys-Pro-Glu-Asp-Asn-His-Asn-Thr-Ser-Phe-Trp-Arg-Asn (Applied Biosystems). For peptide fingerprint mapping and partial peptide sequencing, tryptic peptides were obtained by in-gel digestion in 25 mM NH₄HCO₃ buffer (pH 8.0) for 4 h at 37°C. The digested peptides were extracted from the gel with 5% trifluoroacetic acid/50% acetonitrile. Extracts were lyophilized and resuspended in 1% formic acid for matrix-assisted laser desorption ionization mass spectrometry (MS) and quadrupole time-of-flight (Q-TOF) II MS analysis (Micromass; Waters). One peptide was determined as Leu-Tyr-Ser-His-Ala-Ile-Glu-Glu-Ile-Thr-Lys, matching sequences from Bacillus licheniformis Pz peptidase (1) and Bacillus subtilis PepF (8). A forward primer, 5'-GA(A/G)AA(A/G)CCNGA(A/G)GA(T/C)AA(T/C)CAC-3', and a backward primer, 5'-GT(A/G/C)AT(T/C)TC(T/C)TC(A/G/T)ATNGC(A/G)TG-3', were designed based on N-terminal and internal sequences. The PCR consisted of 30 cycles with an annealing temperature of 47°C, and the product was around 350 bp. Using this amplified fragment as a probe, the complete gene of pepF_Ba was cloned from a B. amyloliquefaciens 23-7A genomic library. PepF_Ba was 2,010 bp long and encoded a protein of 670 amino acid residues. The molecular mass and pI were calculated as 77,049 Da and 5.58 using the ExPASy molecular biology server (http://tw.expasy.org/). A 23-residue signal peptide was defined by the SignalP prediction server (www.cbs.dtu.dk/services/SignalP) (5). Since the first residue of the mature protein was Ser³¹, Ala²⁴-Tyr-Asp-Leu-Thr-Lys-Gly³⁰ might be considered a prosequence that will be removed after the enzyme is secreted. The peptide sequence determined by Q-TOF II MS was found at Leu²⁰²-Lys²¹². The characteristic sequence of a zinc-binding motif, His-Glu-X-X-His, was identified at amino acid positions 456 to 460. PepF_Ba has 87% and 85% identity with B. licheniformis N22 Pz peptidase (1) and B. subtilis PepF (8), respectively (Fig. 2). Pz peptidase is a dimeric metallopeptidase that does not hydrolyze proteins (2). B. subtilis PepF was identified as the homologue of PepF1 in Lactococcus lactis, a well-characterized enzyme that belongs to the M3 family of oligopeptidases (7, 8, 11).

View larger version (105K): [in this window] [in a new window]

FIG. 2. Alignments of the amino acid sequences of PepFBa from B. amyloliquefaciens 23-7A [PepF(BAL)_Bamy] with those of other oligopeptidases of the M3 family. The conserved residues are shown in white on a black background. The zinc binding motif is indicated by asterisks. The signal peptide of PepFBa is boxed, and the first residue of mature protein is indicated by an arrowhead. The internal peptide sequence resolved by matrix-assisted laser desorption ionization-time-of-flight MS is shown by an arrow. The sequences are from B. subtilis PepF (NCBI accession no. CAB13011, PepF_Bsub) (8), B. licheniformis Pz peptidase (NCBI accession no. BAA13561, Pz-pep_Blich) (1), Streptococcus PepB (NCBI accession no. AAC44215, PepB_Strepto) (9), and L. lactis PepF1 (NCBI accession no. CAA83534, PepF1_Lacto) (11).

Enzymatic properties.
The amount of zinc in PepF_Ba was determined by inductively coupled plasma mass spectrometry (7500s; Agilent Technologies). An average of 14.6 ± 0.35 pmol/ml Zn²⁺ was obtained for every 7.8 pmol/ml PepF_Ba, given a molar ratio of 1.87 (Zn²⁺/PepF_Ba). To further determine the enzymatic properties of the proteins under the effects of various chemical reagents as well as temperature and pH, the synthetic peptide FALGPA was used as a substrate to monitor the activity of PepF_Ba. EDTA (10 mM) and 1,10-phenanthroline (10 mM) strongly inhibited PepF_Ba activity, but epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64), iodoacetamide, phosphoramidon, and phenylmethylsulfonyl fluoride did not. Like other bacterial PepFs, PepF_Ba was inhibited by the presence of 10 mM Cd²⁺, Cu²⁺, Fe²⁺, and Pb²⁺ while Ca²⁺ and Mg²⁺ were able to increase PepF_Ba activities. Ca²⁺, Mg²⁺, and Co²⁺ could also cause the restoration of enzyme activity after EDTA treatment (see Table S2 in the supplemental material). PepF_Ba was most active at pH 7. It showed the highest activity at 45°C and became thermally inactive at temperatures over 55°C (see Fig. S2 in the supplemental material). At optimal pH, PepF_Ba was stable at 40°C for 1 h.

Substrate specificity.
The oligopeptide substrates shown in Table 1 were incubated with the enzyme in a ratio of 1/100 (wt/wt) in 20 mM Tris-HCl (pH 7.0) containing 10 mM Ca²⁺ at 37°C. The reactions were stopped by addition of 10 mM EDTA, and the mixtures were subjected to reverse-phase high-performance liquid chromatography using a Cosmosil 5C18-MS column. In the mobile phase, acetonitrile was increased from 0 to 40% of acetonitrile in 40 min, and the eluent was monitored at a wavelength of 214 nm. The recovered eluent was lyophilized, redissolved in water, and then analyzed by Q-TOF II MS (Micromass; Waters). The results indicated that only peptides ranging from 5 to 21 residues in length are cleavable substrates of PepF_Ba. While PepF_Ba revealed no hydrolytic activities on bradykinin residues 1 to 5 (five residues), the substrates FALGPA, 4-phenylazobenzyloxycarbonyl-L-Pro-L-Leu-Gly-L-Pro-D-Arg (Pz peptide), and N-carboxylbenzoyl-Gly-Pro-Leu-Gly-Pro that are 5 residues long were hydrolyzed at a high rate. For FALGPA, the specific activity of PepF_Ba was 20.7 U/mg, and the K_m and k_cat/K_m values were 9.92 x 10^–5 M and 3.3 x 10⁶ M^–1 s^–1, respectively. The cleavage pattern of these substrates by PepF_Ba was similar to that of bacterial PepFs, including L. lactis PepF1 (11) and Streptococcus agalactiae PepB (9). Nevertheless, analysis of the cleavage sites suggested that the bonds cleaved by PepF_Ba are more flexible. For most oligopeptidases in the M3 family, the residues in the P1 position of the cleavage site are mainly hydrophobic and basic ones (3, 10, 16). But PepF_Ba also cleaved acidic residues at the P1 site, such as Glu¹⁷ in oxidized insulin chain A and Glu⁴ in neurotensin.

View this table: [in this window] [in a new window]

TABLE 1. Sites of cleavage of various substrates by PpFBa from B. amyloliquefaciens 23-7Ab

Because of its substrate specificity, sequence similarity, and requirement for Zn²⁺ as a cofactor for enzymatic activity, PepF_Ba should be assigned to the M3 peptidase family. In this family, PepF_Ba and the Pz peptidase from B. licheniformis are secreted proteins, while others are all cytoplasmic. Thus, these extracellular peptidases should play distinct roles from canonical bacterial PepFs. PepF_Ba has sequence similarity with B. subtilis PepF, an enzyme that hydrolyzes intracellular Phr pentapeptides. Phr preproteins are exported outside of cells during the exponential growth phase (6, 18). After processing, active Phr pentapeptides were imported into cells to stimulate the formation of spores (13, 15). The characteristics of Phr pentapeptides are an Arg or Lys in the second residue as well as aliphatic or negatively charged residues in the first position (12). As demonstrated in this study, PepF_Ba is produced extracellularly during the exponential phase. Furthermore, it possesses flexibility in the P1 position and ability to digest substrates with aliphatic residues in the P1' site and Arg or Lys in the P2' site, such as Phe⁷-Arg⁸ and Gly¹⁰-Lys¹¹ (adrenocorticotropic hormone fragment 1-14). It is plausible that PepF_Ba could facilitate sporulation by processing a pro-Phr into the active pentapeptides. On the other hand, PepF_Ba may also play a role in the degradation of peptides that are produced by the action of other secreted proteases. Further studies will be necessary to define its physiological functions.

Nucleotide sequence accession number.
The nucleotide sequence of the B. amyloliquefaciens 23-7A PepF_Ba gene and its encoded amino acid sequence have been deposited in the GenBank nucleotide database under the accession number AF525011.

 
* Corresponding author. Mailing address: Institute of Biochemistry, National Yang-Ming University, 155, Sec. 2, Li-Nong Street, Pei-Tou, Taipei 11221, Taiwan. Phone: (886) 2-2826-7125. Fax: (886) 2-2826-4843. E-mail: tsaiyc{at}ym.edu.tw

Supplemental material for this article may be found at http://aem.asm.org/.

Top Abstract Introduction References

 

1
1. Akiyama, K., K. Mori, and R. Takata. 1999. Cloning and sequencing of the Pz-peptidase gene from Bacillus licheniformis N22. J. Biosci. Bioeng. 87:231-233.[CrossRef][Medline]
2
2. Asdornnithee, S., E. Himeji, K. Akiyama, T. Sasaki, and R. Takata. 1995. Isolation and characterization of Pz-peptidase from Bacillus licheniformis N22. J. Ferment. Bioeng. 79:200-204.[CrossRef]
3
3. Barrett, A. J., M. A. Brown, P. M. Dando, C. G. Knight, N. McKie, N. D. Rawlings, and A. Serizawa. 1995. Thimet oligopeptidase and oligopeptidase M or neurolysin. Methods Enzymol. 248:529-556.[Medline]
4
4. Barrett, A. J., and N. D. Rawlings. 1992. Oligopeptidases, and the emergence of the prolyl oligopeptidase family. Biol. Chem. Hoppe-Seyler 373:353-360.[Medline]
5
5. Bendtsen, J. D., H. Nielsen, G. von Heijne, and S. Brunak. 2004. Improved prediction of signal peptides: SignalP 3.0. J. Mol. Biol. 340:783-795.[CrossRef][Medline]
6
6. Grossman, A. D., and R. Losick. 1988. Extracellular control of spore formation in Bacillus subtilis. Proc. Natl. Acad. Sci. USA 85:4369-4373.[Abstract/Free Full Text]
7
7. Kanamaru, K., S. Stephenson, and M. Perego. 2002. Overexpression of the PepF oligopeptidase inhibits sporulation initiation in Bacillus subtilis. J. Bacteriol. 184:43-50.[Abstract/Free Full Text]
8
8. Kunst, F., N. Ogasawara, I. Moszer, A. M. Albertini, G. Alloni, V. Azevedo, M. G. Bertero, P. Bessieres, A. Bolotin, S. Borchert, R. Borriss, L. Boursier, A. Brans, M. Braun, S. C. Brignell, S. Bron, S. Brouillet, C. V. Bruschi, B. Caldwell, V. Capuano, N. M. Carter, S. K. Choi, J. J. Codani, I. F. Connerton, A. Danchin, et al. 1997. The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature 390:249-256.[CrossRef][Medline]
9
9. Lin, B., W. F. Averett, J. Novak, W. W. Chatham, S. K. Hollingshead, J. E. Coligan, M. L. Egan, and D. G. Pritchard. 1996. Characterization of PepB, a group B streptococcal oligopeptidase. Infect. Immun. 64:3401-3406.[Abstract]
10
10. Monnet, V. 1995. Oligopeptidases from Lactococcus lactis. Methods Enzymol. 248:579-592.[Medline]
11
11. Monnet, V., M. Nardi, A. Chopin, M. C. Chopin, and J. C. Gripon. 1994. Biochemical and genetic characterization of PepF, an oligopeptidase from Lactococcus lactis. J. Biol. Chem. 269:32070-32076.[Abstract/Free Full Text]
12
12. Perego, M., and J. A. Brannigan. 2001. Pentapeptide regulation of aspartyl-phosphate phosphatases. Peptides 22:1541-1547.[CrossRef][Medline]
13
13. Perego, M., and J. A. Hoch. 1996. Cell-cell communication regulates the effects of protein aspartate phosphatases on the phosphorelay controlling development in Bacillus subtilis. Proc. Natl. Acad. Sci. USA 93:1549-1553.[Abstract/Free Full Text]
14
14. Rawlings, N. D., and A. J. Barrett. 1995. Evolutionary families of metallopeptidases. Methods Enzymol. 248:183-228.[Medline]
15
15. Solomon, J. M., B. A. Lazazzera, and A. D. Grossman. 1996. Purification and characterization of an extracellular peptide factor that affects two different developmental pathways in Bacillus subtilis. Genes Dev. 10:2014-2024.[Abstract/Free Full Text]
16
16. Tisljar, U. 1993. Thimet oligopeptidase—a review of a thiol dependent metallo-endopeptidase also known as Pz-peptidase endopeptidase 24.15 and endo-oligopeptidase. Biol. Chem. Hoppe-Seyler 374:91-100.[Medline]
17
17. Van Wart, H. E., and D. R. Steinbrink. 1981. A continuous spectrophotometric assay for Clostridium histolyticum collagenase. Anal. Biochem. 113:356-365.[CrossRef][Medline]
18
18. Waldburger, C., D. Gonzalez, and G. H. Chambliss. 1993. Characterization of a new sporulation factor in Bacillus subtilis. J. Bacteriol. 175:6321-6327.[Abstract/Free Full Text]

---

Applied and Environmental Microbiology, January 2006, p. 968-971, Vol. 72, No. 1
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.1.968-971.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Supplemental material Supplemental material Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Chao, S.-H. Articles by Tsai, Y.-C. Search for Related Content PubMed PubMed Citation Articles by Chao, S.-H. Articles by Tsai, Y.-C. Agricola Articles by Chao, S.-H. Articles by Tsai, Y.-C.