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

ABSTRACT 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 PepFBa oligopeptidase is a secreted protein and may facilitate the process of sporulation.

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 PepFlike 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, grampositive, 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% K 2 HPO 4 , and 0.2% MgSO 4 ) 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 sulfatepolyacrylamide 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 Pep-F Ba forms a homodimeric molecule (see Fig. S1 in the supplemental material).
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 NH 4 HCO 3 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, 5Ј-GA(A/G)AA(A/G)CCNGA(A/G)GA(T/C)AA(T/ C)CAC-3Ј, and a backward primer, 5Ј-GT(A/G/C)AT(T/C)T C(T/C)TC(A/G/T)ATNGC(A/G)TG-3Ј, 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 Ser 31 , Ala 24 -Tyr-Asp-Leu-Thr-Lys-Gly 30 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 Leu 202 -Lys 212 . 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) 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).
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 Ϯ 0.35 pmol/ml Zn 2ϩ was obtained for every 7.8 pmol/ml PepF Ba , given a molar ratio of 1.87 (Zn 2ϩ /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 1,10phenanthroline (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 Cd 2ϩ , Cu 2ϩ , Fe 2ϩ , and Pb 2ϩ while Ca 2ϩ and Mg 2ϩ were able to increase PepF Ba activities. Ca 2ϩ , Mg 2ϩ , and Co 2ϩ 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.
FIG. 1. PepF Ba 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 (OE) determination, the cultured broth was heated for 20 min at 65°C and then spread at appropriate dilutions on LB agar plates. PepF Ba activity (E) was determined by hydrolysis of the peptide FAL-GPA as described previously (17). 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 Ca 2ϩ at 37°C. The reactions were stopped by addition of 10 mM EDTA, and the mixtures were subjected to reverse-phase highperformance 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 ϫ 10 Ϫ5 M and 3.3 ϫ 10 6 M Ϫ1 s Ϫ1 , 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 Glu 17 in oxidized insulin chain A and Glu 4 in neurotensin.
Because of its substrate specificity, sequence similarity, and requirement for Zn 2ϩ 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 P1Ј site and Arg or Lys in the P2Ј site, such as Phe 7 -Arg 8 and Gly 10 -Lys 11 (adrenocorticotropic hormone fragment [1][2][3][4][5][6][7][8][9][10][11][12][13][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 Gen-Bank nucleotide database under the accession number AF525011.