Biochemical characterization of a novel metalloendopeptidase from Streptomyces aureofaciens TH-3 with post-proline hydrolysis activity

Enzyme and Microbial Technology 44 (2009) 295–301

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Biochemical characterization of a novel metalloendopeptidase from Streptomyces aureofaciens TH-3 with post-proline hydrolysis activity Tadashi Hatanaka ∗ , Yoshiko Uesugi, Jiro Arima 1 , Hirokazu Usuki, Masaki Iwabuchi Research Institute for Biological Sciences (RIBS), Okayama, 7549-1 Kibichuo-cho, Kaga-gun, Okayama 716-1241, Japan

a r t i c l e

i n f o

Article history: Received 20 September 2008 Received in revised form 19 November 2008 Accepted 22 November 2008 Keywords: Metalloendopeptidase Streptomyces FRETS

a b s t r a c t Streptomyces aureofaciens TH-3 secretes a protease termed ‘kibilysin’, for which we showed unique substrate specificity and preference for Tyr, Pro, and Leu at the P1 position using fluorescence energy transfer substrate (FRETS) combinatorial libraries. Using (7-methoxycoumarin-4-yl) acetyl-Lys-Pro-Leu-Gly-Leud-2,3-diamino propionic acid (2,4-dinitrophenyl)-Ala-Arg-NH2 , we confirmed that kibilysin digests the substrate between Pro and Leu. Its gene was cloned and sequenced. The primary structure of the enzyme showed 40, 66, and 61% identity, respectively, with those of thermolysin from Bacillus thermoproteolyticus, and metalloendopeptidases from Streptomyces cinamoneus TH-2 and S. griseus. Its deduced amino acid sequence contained an HEXXH consensus sequence for zinc binding, which is a common motif of the peptidase family M4. Moreover, we succeeded in over-expression of kibilysin using Streptomyces lividans. © 2008 Elsevier Inc. All rights reserved.

1. Introduction Proteolysis reactions play an important role in the development of flavor in protein-rich foods. Short peptides and free amino acids play important roles in the induction of umami flavor by eliciting characteristic tastes of food. In contrast, long peptides are generally bitter. Consequently, the extensive hydrolysis of proteins is effective for debittering [1]. Among the 20 amino acids which occur naturally, proline is unique because of its unusual cyclic structure. Peptide bonds involving proline residues are notoriously difficult to cleave using available enzymes. Casein, gluten, collagen, and gelatin are known proline-rich proteins. Their hydrolysates produced by peptidases are limited in their use in food industries because of the property described above. An endopeptidase with a post-proline hydrolysis activity is therefore necessary for the treatment of such proline-rich proteins. Recently, we found that Streptomyces (S.) cinnamoneus (formerly called S. septatus) TH-2 secretes a large amount of a metalloendopeptidase (SCMP) when cultured on a medium containing K2 HPO4 and glucose [2]. Therefore, we decided to screen for proteolysis activity toward fluorescein-conjugated (FITC) gelatin among the streptomycetes available in our laboratory to obtain a protease with a post-proline hydrolysis activity.

∗ Corresponding author. Tel.: +81 866 56 9452; fax: +81 866 56 9454. E-mail address: [email protected] (T. Hatanaka). 1 Present address: Department of Agricultural, Biological, and Environmental Sciences, Faculty of Agriculture, Tottori University, 4-101 Koyama-Minami, Tottori 680-8553 Japan. 0141-0229/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2008.11.005

In this study, we describe the characterization, cloning, sequencing and expression of a metalloendopeptidase from S. aureofaciens TH-3 termed ‘kibilysin’, whose name is derived from the S. aureofaciens TH-3 that was isolated from the ‘Kibi Plateau’ of Okayama prefecture in Japan. Furthermore, the substrate specificity of the enzyme using fluorescence energy transfer substrate (FRETS). 2. Materials and methods 2.1. Materials FITC-gelatin and FRETS were purchased from Molecular Probes Inc. and Peptide Institute Inc., respectively. Thermolysin was obtained from Wako Pure Chemicals Industries Ltd. 2.2. Culture S. aureofaciens TH-3 was grown in 50 ml of a culture medium containing 2.0% glucose, 0.8% K2 HPO4 , 0.05% MgSO4 ·7H2 O, 0.5% polypeptone and 0.5% yeast extract in a 500-ml baffled flask at 30 ◦ C for 3 days with rotary shaking at 125 rpm. 2.3. Kibilysin purification All experiments were performed at 4 ◦ C unless otherwise noted. A culture filtrate was obtained using an ultrafiltration apparatus (0.45 ␮m pore size; Millipore Corp.). The filtrate was concentrated through ammonium sulfate precipitation at 70% saturation. The precipitates, which were collected by centrifugation at 15,000 × g for 2 h, were dissolved in 20 mM Tris–HCl buffer (pH 8.0) containing 0.2 M NaCl. The sample was loaded onto a HiLoad 16/60 Superdex200 column (GE Healthcare Bio-Science) equilibrated with the buffer mentioned above. The active fractions exhibiting high activities were pooled and dialyzed against 20 mM Tris–HCl (pH 8.0) buffer. After centrifugation, the supernatant was loaded onto a Mono Q HR5/5 column that had been pre-equilibrated with the dialysis buffer. The enzyme was eluted using a linear gradient of NaCl from 0 to 0.25 M using 20 bed volumes of the same buffer. The obtained enzyme solution was used for characterization.

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Fig. 1. (A) Structure of FRETS-25Xaa combinatorial libraries. Xaa denotes the fixed position at which each of the 19 natural amino acids excluding Cys was incorporated. A mixture of five amino acid residues (P, Y, K, I, and D) was incorporated at the Yaa position along with a mixture of five amino acid residues (F, A, V, E, and R) at the Zaa position for each fixed Xaa. Determination of substrate specificity of kibilysin using FRETS-25Xaa combinatorial libraries. (B) Primary screening at P1 position using FRETS-25Xaa combinatorial libraries. (C) Secondary screening at P1 , P2 and P3 positions using FRETS-25Leu. Relative fluorescence intensity was determined from the peak area by LC–MS. Cleavage sites are represented as a slash.

2.4. FITC-gelatin hydrolysis assay The FITC-gelatin was heavily labeled with fluorescein such that the fluorescence was quenched when it was digested by proteases to detect its fluorescence. The FITC-gelatin was dissolved in distilled water (1 mg/ml). Then 10 ␮l of the substrate solution was added to 180 ␮l of 100 mM Tris-maleic acid buffer (pH 7.0) containing 25 mM CaCl2 per well of a microtiter plate for fluorescence analysis. To start the reaction, 10 ␮l of the enzyme solution was added to each well. The reaction was monitored in terms of the increase in fluorescence intensity at ex 535 nm and em 485 nm using a multilabel counter (ARVO 1420; PerkinElmer Inc.) at 37 ◦ C. The reaction velocity was estimated from the standard curve plotted using fluorescein isothiocyanate (Wako Pure Chemical Industries Ltd.) solution. Purification and biochemical characterization of kibilysin were performed according to the hydrolysis activity of FITC-gelatin. 2.5. Analysis of FRETS combinatorial libraries In fact, FRETS-25Xaa libraries (Fig. 1a) contain a highly fluorescent 2-(Nmethylamino)benzoyl (Nma) group linked to the side chain of an amino terminal d-2,3-diamino propionic acid (A2 pr) residue, which is quenched efficiently by a 2,4dinitrophenyl (Dnp) group linked to the ␧-amino function of Lys. We performed an enzyme reaction using 100 mM Tris-maleate buffer (pH 7.0) at 37 ◦ C. We used 50 ng of purified kibilysin for the primary screening. The procedure using FRETS followed that of Hatanaka et al. [2]. The reaction was monitored in terms of the increase in fluorescence intensity at ex 355 nm and em 460 nm using the multilabel counter (ARVO1420; PerkinElmer Inc.). For the secondary screening, we chose FRETS-25Leu as the best substrate among the libraries. We performed an enzyme reaction using 200 ng of purified kibilysin at 37 ◦ C for 7 or 14 min to determine the preference of the enzyme for P2 and P3 positions. We added EDTA to the reaction mixture to stop the reaction; then we determined the digestion rate of the substrate. The amount of the resultant sample was digested by approximately 5–17%. We requested that the Peptide Institute Inc. identify the sequences derived from FRETS-25Leu digested using purified kibilysin by liquid chromatography (LC)–mass spectrometry (MS).

KPLGL-Dnp [3], Peptide Institute Inc.) was used for the kinetic study of kibilysin based on results obtained using FRETS-25Xaa libraries. Stock solutions of the substrate were diluted with 100 mM Tris-maleate buffer (pH 7.0) containing 25 mM CaCl2 . The diluted substrate (3–50 ␮M, 490 ␮l) was preincubated for 5 min at 37 ◦ C. Then, 10 ␮l of the purified kibilysin (50 ng/␮l) was added to the substrate solution; which was then monitored during 0–30 s at 37 ◦ C. The assay was performed using a fluorescence spectrophotometer (F-4500; Hitachi Ltd.) with a thermal cell holder (excitation at 328 nm and emission at 393 nm). The initial velocity was estimated from the standard curve drawn using MOCAc-Pro-Leu-Gly (Peptide Institute Inc.) solution. 2.7. High performance liquid chromatography (HPLC) analysis We performed HPLC using an HPLC millennium system with a scanning fluorescence detector (474; Waters Corp.) and a 150 mm × 4.6 mm column (Hydrosphere C18; YMC Co. Ltd.). Eluent A was 0.1% (v/v) trifluoroacetic acid (TFA) and eluent B was water/acetonitrile/TFA, 30/70/0.085% (v/v/v). A gradient from 20 to 100% of eluent B in 20 min at a flow rate of 0.8 ml/min was used. The detection wavelengths were 325 nm (excitation) and 400 nm (emission). Then 25 ␮M of MOCAc-KPLGL-Dnp was dissolved in 0.1 M Tris-maleate (pH 7.0) containing 25 mM CaCl2 . Purified kibilysin (100 ng) was added to 100 ␮l of the substrate solution; then the mixture was incubated at 37 ◦ C for 5 min. After heat treatment (at 90 ◦ C for 5 min) to stop the enzyme reaction, an equal volume of water/acetonitrile/TFA, 70/30/0.1% (v/v/v) was added to the sample, and 10 ␮l of this mixture was used for HPLC analyses. We requested that Waters Corp. identify the molecular mass of a fragment from MOCAc-KPLGL-Dnp digested with kibilysin by LC–MS. 2.8. Matrix assisted laser desorption ionization (MALDI) time-of-flight mass (MALDI-TOF-MS) analysis Samples were purified using solid-phase extraction (ZipTip ␮C18; Millipore Corp.) and were subsequently subjected to MALDI-TOF-MS analysis (Autoflex II TOF/TOF; Bruker Daltonics Inc.). 2.9. Gel filtration analysis

2.6. Kinetics study The FRETS containing a highly fluorescent (7-methoxycoumarin-4-yl)acetyl (MOCAc) group, MOCAc-Lys-Pro-Leu-Gly-Leu-A2 pr(Dnp)-Ala-Arg-NH2 (MOCAc-

Gel filtration was performed using a Superdex 200 10/300 column (GE Healthcare Bio-Science) that had been pre-equilibrated with 20 mM Tris–HCl (pH 8.0) containing 0.2 M NaCl. Molecular weight marker proteins (glutamate dehydrogenase

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[290 kDa], lactate dehydrogenase [142 kDa], enolase [67 kDa], myokinase [32 kDa], and cytochrome C [12.4 kDa]) were purchased from Oriental Yeast Co. Ltd. 2.10. Determination of N-terminal and internal amino acid sequences The purified kibilysin was electroblotted and then loaded onto the protein sequencer to identify its N-terminal amino acid sequence (AAGTGNVFV). Internal sequences (ESAAVDAQFG and AATLSAASDLYG) were obtained by digesting the enzyme with trypsin. We also confirmed that the secreted recombinant kibilysin possessed the same N-terminal amino acid sequence as that of the wild type. We requested that APRO Science Co. Ltd. determine the N-terminal and internal sequences. 2.11. Cloning of kibilysin gene using inverse polymerase chain reaction (PCR) Genomic DNA was prepared from S. aureofaciens TH-3 using the methods of Hopwood et al. [4]. The kibilysin gene fragment was amplified by PCR from genomic DNA using the generated primers (forward 5 -AC(CG)GG(CG)AACGG(CG)GT(CG)TTCGT3 and reverse 5 -CCGTA(CG)A(GA)GTC(CG)(CG)(TA)(CG)GC(CG)GC-3 ), which were designed from the N-terminal (AAGTGNVFV) and internal (AATLSAASDLYG) sequences of kibilysin. Actually, PCR was carried out for 30 cycles of 30 s at 98 ◦ C, 30 s at 50 ◦ C, and 1 min at 72 ◦ C, proceeded by incubation of 1 min at 98 ◦ C and followed by incubation of 5 min at 72 ◦ C. The PCR product was cloned into the pGEM-T easy vector (Promega Corp.) and sequenced. A digoxigenin (DIG)-labeled probe was synthesized using part of the kibilysin gene sequence and a PCR DIG probe synthesis kit (Roche Molecular Biochemicals). This probe was hybridized to 2.5 kb fragments of genomic DNA digested using BalI. Next, these fragments were recovered using agarose gel electrophoresis and self-ligated. The ligation products were amplified using primer sets designed from the partial DNA fragment of the kibilysin gene. The PCR products were cloned and sequenced. The sequence has been assigned accession number AB281185 in the DDBJ database. 2.12. Construction of kibilysin expression vectors We attempted to obtain a recombinant SCMP by using Escherichia (E.) coli, however, it was not successful by formation of inclusion bodies [2]. Recently, we constructed an expression vector (pTONA5a), which included a promoter from Streptomyces metalloendopeptidase, using an intergenic conjugation between E. coli and streptomycetes [5]. Thus, we planed an expression of kibilysin gene using this vector that was expected to be its extracellular production. For this study, S. lividans 1326 was used as a host strain for kibilysin expression. The kibilysin gene was amplified by PCR using a set of sense primer incorporating the NdeI site (CATATG; the start codon is underlined) and anti-sense primer incorporating the HindIII site downstream of the stop codon and KOD ver.2 DNA polymerase (Toyobo Co. Ltd.). The resultant fragment was cloned and sequenced using a cloning vector (pCR-Blunt II-TOPO; Invitrogen Corp.). The DNA fragment encoding kibilysin was digested with NdeI and HindIII, and ligated into the NdeI–HindIII gap of pTONA5a (pTONA5kibilysin). 2.13. Expression and preparation of a recombinant An expression vector was transformed in E. coli S17-1. A single colony of the transformant was cultivated using Luria Bertani (LB) broth containing 50 ␮g/ml kanamycin (Km) at 37 ◦ C for 7 h. Cells were harvested and washed with LB broth by pelleting the cells three times to remove Km. The cells were suspended in 500 ␮l of LB broth and then mixed with spores of S. lividans 1326. The mixture was spread on an ISP No. 4 agar plate and incubated at 30 ◦ C overnight. A 3 ml aliquot of softagar nutrient broth containing Km (50 ␮g/ml) and nalidixic acid (Nal, 67 ␮g/ml) was dispensed as layers on the plate, which was then incubated at 30 ◦ C for 3–5 days. A single colony was streaked on an agar plate with a medium containing 2.0% soybean meal, 2.0% mannitol, Km (20 ␮g/ml), and Nal (5 ␮g/ml) in tap water, which was autoclaved twice. The plate was incubated at 30 ◦ C for 5–7 days. The resultant S. lividans 1326 transformants were inoculated and grown in 50 ml of a culture medium containing 0.8% K2 HPO4 , 2.0% glucose, 0.05% MgSO4 ·7H2 O, 0.5% polypeptone, and 0.5% yeast extract (PG medium) in a 500-ml baffled flask at 30 ◦ C for 6 days with rotary shaking at 180 rpm [5]. A culture filtrate was obtained using an ultrafiltration apparatus (0.45 ␮m pore size; Millipore Corp.). The filtrate was desalting by PD-10 column pre-equilibrated with 10 mM Tris-maleate (pH 7.0). A little loss of activity was observed by desalting comparing to that of the culture filtrate. The resultant

Fig. 2. (A) SDS-PAGE of purified kibilysin. Lane 1 contained molecular weight markers. Lanes 2, 3, 4 and 5 contained 3 ␮g of the culture supernatant, the filtrate concentrated through ammonium sulfate precipitation at 70% saturation, the active fraction of gel filtration and the active fraction of Mono Q. (B) SDS-PAGE of r-kibilysin. Lane 1 contained molecular weight markers. Lane 2 contained 3 ␮g of the recombinant. sample was only included the recombinant protein on SDS-PAGE (Fig. 2B), therefore, used for the experiments.

3. Results 3.1. Purification and biochemical characterization of kibilysin To determine kibilysin characteristics, we first purified kibilysin from the culture supernatants of S. aureofaciens TH-3; the enzyme purification procedure is presented in Table 1. From 150 ml of culture filtrate, the enzyme was purified to homogeneity (Fig. 2A), resulting in a 13.1-fold purification of kibilysin in 8.0% yield (Table 1). Kibilysin was determined to be a monomer based on SDSPAGE (Fig. 2A), gel filtration analysis, and MALDI-TOF-MS analysis (data not shown). The basic enzymological properties such as optimum pH and temperature, thermal and pH stabilities, activators and inhibitors are summarized in Table 2. Metalloendopeptidases from Bacillus and Streptomyces are stabilized by calcium ions [2,6]. Consequently, we examined the effects of calcium on kibilysin activity and stability. The activity was enhanced (1.3-fold) by 25 mM calcium, but not its stability (data not shown). An enhancement effect (1.7-fold) by CoCl2 at 10 mM in the presence of 25 mM CaCl2 was also apparent (data not shown). Interestingly, the activity of kibilysin was inhibited by dithiothreitol in addition to EDTA and 1,10-phenanthroline. For that reason, we examined the effects of reduced and oxidized glutathiones on kibilysin activity. In the presence of 10 mM reduced and oxidized of glutathiones, the enzyme activity was also inhibited completely.

Table 1 Kibilysin purification. Fraction

Activity (U/ml)

Protein (mg/ml)

Specific activity (U/mg)

Total volume (ml)

Total unit (U)

Yield (%)

Purity (-fold)

Supernatant 70% (NH)4 SO4 precipitation Gel filtration Mono Q

0.19 4.81 1.04 1.11

0.48 7.00 0.42 0.21

0.39 0.69 2.48 5.26

150.0 7.0 11.0 2.0

28.0 33.6 11.4 2.2

100.0 120.2 40.9 8.0

1.0 1.8 6.2 13.1

Enzyme activity was determined using 50 ␮g/ml FITC-gelatin in 80 mM Tris-maleate (pH 7.0) containing 25 mM CaCl2 at 37 ◦ C. One unit of the enzyme activity was determined as the amount of enzyme required to release 1 nmol of FITC per minute under the conditions described above.

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Table 2 Biochemical properties of kibilysin.

3.2. Nucleotide sequence of kibilysin gene

Property

Purified kibilysin

Molecular mass of promoter Composition Optimum pH Optimum temperature Thermal stability Activators Inhibitors

34 kDa Monomer 6.5–7.3 40 ◦ C 50 ◦ C Ca2+ and Co2+ EDTA, 1,10-phenanthroline, dithiothreitol and glutathione

Enzyme activity was determined using 50 ␮g/ml FITC-gelatin in 80 mM Tris-maleate (pH 7.0) at 37 ◦ C.

Fig. 3 shows the nucleotide sequence of the kibilysin gene. From the N-terminal and internal amino acid sequences analyses, there was an in-frame stop codon, TAG, at nucleotide (nt) 1861–1863. The primary structure of kibilysin shows 40% (76%), 66% (93%) and 61% (90%) identities (similarities) with those of thermolysin from Bacillus thermoproteolyticus [9], SCMP [2] and SGMP [7], respectively (Fig. 4). Comparison of the kibilysin gene with those from the two Streptomyces metalloendopeptidases seemed to show that kibilysin possesses an N-pre sequence (11 amino acid residues) and an N-pro sequence (209 amino acid residues). The open reading frame was predicted to be 1644 nt long and to encode a kibilysin enzyme of 547 amino acids with a predicted molecular mass of 56945. The calculated molecular mass of the mature kibilysin enzyme is 34389.

Fig. 3. Nucleotide sequence of kibilysin gene. Probable −35, −10 sequences and a ribosome-binding (SD) sequence are under lined. The consensus sequence (HEXXH) for zinc binding was boxed. A possible cleavage site (open triangle) of a signal peptidase is shown. During kibilysin maturation, secondary cleavage (the site is indicated by a closed triangle) probably occurred after secretion [8]. In the primary structure deduced from amino acid sequence, N-terminal (underlined) and internal (double-underlined) sequences were found.

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Fig. 4. Alignment of primary structures of mature metalloendopeptidases. SCMP is a metalloendopeptidase from S. cinamoneusTH-2. SGMP is metalloendopeptidase II from S. griseus, and thermolysin is from Bacillus thermoproteolyticus. Asterisks denote identical residues among the four sequences. The consensus sequence (HEXXH) for the zinc-binding site is underlined and the CFC motif is boxed. Phe-114 of thermolysin and Phe residues corresponding to that in kibilysin, SCMP and SGMP are highlighted in black.

Its deduced amino acid sequence contained an HEXXH consensus sequence for zinc binding, confirming that it encodes metalloendopeptidase, which belonged to the peptidase family M4 by the MEROPS database. 3.3. Substrate preference of kibilysin using FRETS-25Xaa libraries When the libraries (475 peptide substrates, Fig. 1A) were used for primary screening, FRETS-25Leu was the best substrate among them to investigate the substrate preference (Fig. 1B). Therefore, FRETS-25Leu was chosen as the substrate for the secondary screening. As portrayed in Fig. 1C, kibilysin had a P1 preference for Tyr, Pro, and Leu, a P2 preference for Arg, Ala, Phe, and Tyr, and a P1 preference for Leu and Ala. The results reveal that kibilysin clearly has post-proline hydrolysis activity. Kibilysin showed a low activity toward FRETS-25Pro that has a Gly-Zaa-Yaa-Pro-Ala-Phe sequence. Therefore, its post-proline hydrolysis activity seemed to be affected by the residues at P3 , P2 , and P1 positions. 3.4. Confirmation of the post-proline hydrolysis activity We chose a FRETS (MOCAc-KPLGL-Dnp) designed for matrix metalloendopeptidases [3] because the substrate possessed ProLeu in the sequence. We performed HPLC analysis (Fig. 5A) to confirm the post-proline hydrolysis activity of kibilysin. By diges-

tion with kibilysin, a new peak was detected at a retention time of 6 min. The fluorescence intensity of this peak increased 10 times more than that of an intact substrate (MOCAc-KPLGL-Dnp), which was detected at retention time of 17 min. We confirmed that the digested substrate included fragments of MOCAc-KP and LGL-Dnp using LC–MS (data not shown) and MALDI-TOF-MS (Fig. 5B). The averaged molecular mass corresponding to LGLDnp is 779.848, which was detected using MALDI-TOF-MS (mass number: 780.292, Fig. 5B). Two nitro groups of LGL-Dnp are thought to be reduced by laser irradiation of MALDI-TOF-MS; one oxygen atom or two atoms were thereby lost. By MALDITOF-MS analysis, two peaks corresponding to mass numbers of 764.166 (One oxygen atom was lost.) and 748.100 (Two atoms were lost.) were detected (Fig. 5B). According to the results described above, kibilysin is inferred to have post-proline hydrolysis activity. 3.5. Over-expression of kibilysin The transformants were secreted large amounts of recombinant (r-) kibilysin. The recombinant formed almost of extra cellular proteins (Fig. 2B). The Km , kcat , and kcat /Km of purified kibilysin from S. aureofaciens TH-3 toward MOCAc-KPLGL-Dnp were 28.1 ± 2.5 ␮M, 12.0 ± 0.8 (1/s) and 0.43 ± 0.01 ((1/s) × (1/␮M)), and those of a purified recombinant were, respectively, 34.2 ± 5.9 ␮M, 13.3 ± 1.6 (1/s)

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Fig. 5. (A) Hydrolysis of MOCAc-KPLGL-Dnp by kibilysin, as determined by HPLC/fluorescence detection. The upper line represents 125 nmol of MOCAc-KPLGLDnp. The lower line represents 125 nmol of MOCAc-KPLGL-Dnp digested by kibilysin for 5 min at 37 ◦ C. (B) MALDI-TOF-MS analysis of MOCAc-KPLGL-Dnp digested using kibilysin for 5 min at 37 ◦ C.

kibilysin is probably changed. Recently, we developed a method of random chimera genesis between two homological genes by using E. coli, which was named as RIBS shuffling [19]. Further study is in progress to identify the amino acid residue corresponding to the substrate preference by using RIBS shuffling. From the result using FRETS-25Xaa libraries, the substrate preference of kibilysisn is affected by the residues of P2 , P3 and P1 positions. Moreover, the Km value toward MOCAc-KPLGL-Dnp of kibilysin is ten times higher than those of matrix metalloproteinases 13 and 14 [3]. In consideration of above results, a combination of kibikysin and other peptidases, such as prolyl endopeptidases [20] and/or aminopeptidases [5], is needed to degrade proline-rich proteins completely. Actually, SGMP is also known to be inhibited strongly by protein serine protease inhibitors produced by streptomycetes, such as the S. subtilisin inhibitor (SSI). Kojima et al. described that the Cys-PheCys (CFC) motif in SGMP is present within 10–15 Å of the zinc atom as the active center in the putative tertiary structure of SGMP [21]. Near the zinc atom in the active site of SGMP, the motif, which is conserved among kibilysin, SCMP, and SGMP (Fig. 4), forming an unusual disulfide bond might be a candidate SSI-binding region. Because the activity of kibilysin was inhibited by dithiothreitol and glutathione (Table 2), a disulfide bond between its CFC motif, in which only two Cys residues exist in mature kibilysin, might be formed. Therefore, we speculate that the reduction or modification of the disulfide bond between the CFC motif engenders the inactivation. In conclusion, we purified, characterized, cloned, sequenced, and over-expressed kibilysin from S. aureofaciens TH-3. We demonstrated a Streptomyces metalloendopeptidase with a post-proline hydrolysis activity using FRETS. Its deduced amino acid sequence contained the HEXXH consensus sequence for zinc binding and the CFC motif that conserved among Streptomyces metalloendopeptidases. We also succeed in over-expression of kibilysin using S. lividans. Considering the post-proline specificity and the overproduction of this enzyme, we believe that kibilysin is useful for hydrolysis of hard-to-degrade proteins, such as collagen and gluten, in food industries.

and 0.39 ± 0.03 ((1/s) × (1/␮M)). Consequently, the activity of the recombinant resembles that of the wild type

Acknowledgement

4. Discussion

We thank Dr. M. Tsunemi, Peptide Institute Inc. for helpful advice during this study.

Recently, the substrate preferences of several proteases have been investigated using FRETS combinatorial libraries [2,10–12]. However, no reports have described protease with a post-proline hydrolysis activity using FRETS. The substrate preferences of thermolysin have already been investigated using FRETS combinatorial libraries, but the enzyme does not show a post-proline hydrolysis activity. The order of preference of thermolysin is Phe, Leu, Met, and Tyr at the P1 position [10]. We have also investigated the substrate specificity of SCMP using FRETS combinatorial library [2]. In fact, SSMP prefers Phe, Leu, Tyr, Met, Thr, and Ala residues at the P1 position in descending order. Kajiwara et al. reported that SGMP prefers the cleavage of the Phe-Xaa bond using a bimane peptide, but SGMP with post-proline hydrolysis activity is not examined in their study [13]. According to the MEROPS database, numbers of peptidases can hydrolyze a peptide bond on the C-terminal of Pro. For instance, as for thermolysin, several studies have been described on its postproline hydrolysis [14–17]. As mentioned above, two Streptomyces metalloendopeptidases, kibilysisn and SCMP, were differed in their substrate preferences although their primary structures were similar (Fig. 4). Hangauer et al. described that Phe-114 of thermolysin, in which the residue was conserved among kibilysin, SCMP, SGMP and thermolysin (Fig. 4), was positioned P1 site of Z-Phe-Phe-LeuTrp [18]. We guess that an environment around the Phe residue in

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