Molecular dissection of Streptomyces trypsin on substrate recognition

Biochimica et Biophysica Acta 1814 (2011) 1295–1304

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a p a p

Molecular dissection of Streptomyces trypsin on substrate recognition Yoshiko Uesugi 1, Hirokazu Usuki, Jiro Arima 2, Masaki Iwabuchi, Tadashi Hatanaka ⁎ 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 31 August 2010 Received in revised form 9 June 2011 Accepted 14 June 2011 Available online 13 July 2011 Keywords: Serine protease Substrate recognition Surface plasmon resonance Collagen binding RIBS in vivo DNA shuffling

a b s t r a c t We recently identified residue 71 of two homologous serine proteases from Streptomyces omiyaensis (SOT) and Streptomyces griseus (SGT) as a crucial residue for differences in their topological specificities, i.e. recognition of a distinct three-dimensional structure. To study the role of this key residue in substrate recognition, we used surface plasmon resonance analysis to evaluate the affinities of inactive mutants, in which residues 71 of SOT and SGT were substituted respectively with Leu and Tyr, toward different types of collagens. We identified another amino acid residue involved in the interaction with collagens from analyses of inactive chimeras between SOT and SGT using an in vivo DNA shuffling system. Results showed that residue 72 contributes to collagen binding. By substituting Leu71 and Gln72 with Tyr and Arg, respectively, SGT mutant showed a change in topological specificity and high hydrolytic activity toward type IV collagen comparable to SOT. We demonstrated that the neighboring residues 71 and 72 in the N-terminal β-barrel domain of the enzyme synergistically play an important role in substrate recognition. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Chymotrypsin-like serine proteases are the most abundant in nature, with more than 8880 protease sequences included in the MEROPS database [1]. These proteases are found in eukaryotes, prokaryotes, archae, and viruses. Substrate recognition is a key concept in studies of serine proteases involved in several important physiological functions including protein processing, tissue remodeling, fibrinolysis, cell differentiation, and blood coagulation [2]. In addition, serine proteases have recently received much attention in relation to degradation of recalcitrant animal proteins, such as collagen [3,4], keratin [5], blood clots [6,7], and amyloid prion proteins [8] for beneficial use of industrial waste and medical applications. To date, the structure and the specificity at primary S1–Sn sites of serine proteases have been studied extensively. However, the recognition mechanism of the structural protein substrate in serine proteases remains unclear. Various structural features govern interactions between proteases and substrates. There-

Abbreviations: SOT, Streptomyces omiyaensis serine protease; SGT, Streptomyces griseus trypsin; SPR, surface plasmon resonance; RU, resonance unit; CD, circular dichroism; RIBS, repeat-length independent and broad spectrum ⁎ Corresponding author. Tel.: + 81 866 56 9452; fax: + 81 866 56 9454. E-mail address: [email protected] (T. Hatanaka). 1 Present address: Department of Biomaterials, Field of Tissue Engineering, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawara-cho Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. 2 Present address: Department of Agricultural, Biological, and Environmental Sciences, Faculty of Agriculture Tottori University, 4-101 Koyama-Minami, Tottori 680-8553, Japan. 1570-9639/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2011.06.015

fore, our study is devoted to clarification of the recognition mechanism of structural protein substrates in serine proteases. Very recently, we first found the key amino acid residue responsible for topological specificity using a serine protease from Streptomyces omiyaensis (SOT; the DDBJ accession number AB362837) as a model enzyme [9]. A trypsin family and peptidase family S1 (subfamily S1A) member, SOT has much higher collagenolytic activity than commercial Clostridium histolyticum collagenase. Although SOT shows 77% identity with a trypsin from Streptomyces griseus (SGT, EC 3.4.21.4) [10–12] in the primary structures (SI Fig. 9), they differ in their topological specificity [3]. Moreover, we selected collagens as a structural protein substrate because collagens have many types: types I, II, III, V, and IX are classical fibrillar collagens; and type IV forms sheet-like networks [13,14]. Elucidation of the recognition mechanism of the structural protein substrate including substrate specificity and interaction with substrate is useful for industrial applications and development of therapeutic agents. Recently, real-time surface plasmon resonance (SPR) analysis has been used to study the kinetics of protein adsorption/desorption and surface enzymatic reactions [15]. In other studies, interaction with the collagen-binding domain (CBD) of Clostridium histolyticum collagenase and collagen substrate was analyzed using SPR analysis [16,17]. For this study, to investigate the roles of the key residue conferring topological specificity of substrate recognition in greater detail, we first analyzed the effect of the key residue on interaction with different types of collagens using SPR analysis. In general, the catalytic reaction of serine proteases is a three-step mechanism: formation of an enzyme–substrate complex; acylation of the catalytic Ser; and hydrolysis of the acylenzyme intermediate [18]. Therefore, we constructed inactive mutants of SOT and SGT, in which the key residue on topological specificity was

1296

Y. Uesugi et al. / Biochimica et Biophysica Acta 1814 (2011) 1295–1304

mutated concomitantly with substitution of the catalytic Ser with Ala. Thereby, we determined the interaction for the enzyme–substrate complex before the tetrahedral intermediate via Ser. We identified another amino acid residue(s) related to the collagen binding using inactive chimeras between SOT and SGT with repeat-length independent and broad spectrum (RIBS) in vivo DNA shuffling [19]. Moreover, we investigated the effect of the identified key residues on the hydrolytic activity using site-directed mutagenesis. 2. Materials and methods 2.1. Materials A negatively-charged ion-exchange spin column was purchased from (Vivapure S; Vinascience, Sartorius AG). A protein assay kit and gels for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were purchased (BioRad Laboratories Inc.). In addition, DQ-collagens were purchased (Molecular Probes Inc.). Type I collagen from bovine Achilles tendon, type IV collagen from human placenta, and Clostridium histolyticum collagenase type I were purchased from Sigma-Aldrich Inc. Benzyloxycarbonylglycyl-L-prolyl-L-arginine-4-methylcoumaryl-7amide (Z-Gly-Pro-Arg-MCA) was obtained from Peptide Institute Inc. All other chemicals were of the highest purity available. 2.2. Construction of inactive mutants To investigate effects of the identified residues related to topological specificity on the interaction with collagens, we constructed inactive SOT, SGT, SGT-L71Y and SOT-Y71L mutants in which Ser172 of the catalytic triad was substituted with Ala. To construct inactive SOT, the mutagenic gene was amplified using PCR with the following primers: 5′-GCGTCGACACCTGTCAGGGCGAC(T → G, Ser172 → Ala)CCGG-3′ (a forward primer, corresponding to nt 608– 635 from sot) and 5′-AAGCTTTACAGCGTGGCCGCGGCGG-3′ (a reverse primer, corresponding to the HindI site of sot) using KOD-Plus (ver.2; Toyobo Co. Ltd.). The amplified DNA fragment was cloned into the pCRBlunt II-TOPO (Invitrogen Corp.); the resultant plasmids were confirmed by DNA sequencing. The plasmid was digested with SalI and HindIII. The plasmid pCR-Blunt II-TOPO (SOT) [3] was digested with NdeI and SalI. The fragments were ligated into the NdeI–HindIII gap of pTONA5a [20]; the resulting expression vector pTONA5a(SOT-S172A) was obtained. To construct inactive SGT, the target mutation was introduced with primer sets of 5′-CACCGGTGGCGTCGACACCTGCCAGGGTGAC(T →G, Ser172 → Ala)CC-3′ (a forward primer, corresponding to nt 594–627 from sprT) and 5′-TGCCGGTACGAAGCTTCAGAGCGTGCG-3′ (a reverse primer, corresponding to the HindIII site of sprT). The amplified DNA fragment was then cloned, sequenced and digested with AgeI and HindIII. The plasmid pCR-Blunt II-TOPO (SGT) [3] was digested with NdeI and AgeI. The fragments were ligated into the NdeI–HindIII gap of pTONA5a; the resulting expression vector pTONA5a(SGT-S172A) was obtained. Next, inactive SGT-L71Y and SOT-Y71L mutants were constructed using the consensus-unique Aor51HI site (corresponding to nt 377–382 from sot). The mutants SGT-L71Y and SOT-Y71L were constructed using PCR amplification, as detailed previously [9]. The genes representing SGT-L71Y and SOT-Y71L were digested with NdeI and Aor51HI and ligated into the NdeI-Aor51HI gap of pTONA5a(SGT-S172A) and pTONA5a(SOT-S172A) to construct the expression vectors. 2.3. Expression and purification of enzymes The respective expression of active and inactive SOT, SGT, and their mutants were performed by application of a novel expression system [20] using the expression vector (pTONA5a), which included a promoter from Streptomyces metalloendopeptidase, with S. lividans 1326 as a host strain. An expression vector was transformed in E. coli S17-1. A single colony of the transformant was cultivated using LB

medium containing 50 μg/ml kanamycin (Km) at 37 °C for 8 h. Cells were harvested and washed with LB medium three times to remove Km. The cells were suspended in 500 μl of LB medium and then mixed with spores of S. lividans 1326. After incubation of the mixture on an ISP No. 4 agar plate at 30 °C for 18–22 h, 4.5 ml of soft-agar nutrient broth containing Km (50 μg/ml) and nalidixic acid (Nal, 67 μg/ml) was dispensed as layers on the plate and incubated for an additional 3 days at 30 °C. A single colony was streaked on an agar plate with medium containing 2.0% soybean meal, 2.0% mannitol, Km (20 μg/ml), and Nal (5 μg/ml) in tap water, and incubated at 30 °C for 2 days. The resultant S. lividans 1326 transformants were inoculated and cultivated in 50 ml of a culture medium containing 2% glucose, 0.5% polypeptone, 0.5% yeast extract, 0.8% K2HPO4 and 0.05% MgSO4·7H2O in a 500-ml baffled flask at 30 °C for 6 days with rotary shaking at 180 rpm. The culture supernatant containing active and inactive SOT was dialyzed against 25 mM sodium acetate buffer (pH 5.5) at 4 °C. The sample was loaded on a negatively-charged ion-exchange spin column (Vivapure S) equilibrated with the buffer described above. The bound proteins were eluted with buffer containing 0.5 M NaCl. Purification of active and inactive SGT followed a similar procedure except for the use of 20 mM sodium acetate buffer (pH 4.5). Inactive mutants were purified in a procedure similar to that used for SOT or SGT. The resultant enzyme solutions were dialyzed against 10 mM Tris–HCl (pH 8.0). Then the purities of the proteins were confirmed using SDS-PAGE [21]. The protein concentrations were measured using a protein assay kit (BioRad Laboratories Inc.), which is based on the Bradford method, according to the standard procedure.

2.4. Active site titration To confirm the active site concentrations of SOT and SGT, we performed the titration using methylumbelliferyl p-guanidinobenzoate. The procedure was following Coleman et al. except for using 0.1 M sodium phosphate buffer (pH 6.8) containing 1 mM HCl [22]. The fluorescence intensity was monitored at λex 323 and λem 446 using a CORONA grating microplate reader SH-8000Lab. The active site concentrations were estimated by using a standard curve for the fluorescence of 4-metylumbelliferone. The concentration of SOT and SGT were 40.2 μM and 10.6 μM, when their concentrations that were determined by means of Bradford assay were estimated 56.8 μM and 15.1 μM, respectively. The active forms of SOT and SGT accounted for 70.8% and 70.4% of total protein. From these results, we judged that the amounts of enzymes used in this study were correctly matched.

2.5. SPR analysis Real-time interactions of inactive SOT, SGT, and their mutants with native collagens were measured using surface plasmon resonance with a biosensor instrument (BIAcore 2000; Biacore AB; Uppsala, Sweden). Native type I or IV collagen was covalently coupled via primary amine groups to the dextran matrix of a Sensor Chip CM5 (Biacore AB). After activation of the carboxymethylated dextran layer by addition of 200 μl of a mixture of 50 mM N-hydroxysuccinimide and 200 mM N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide at a flow rate of 10 μl/min, 200 μl of 0.1 mg/ml collagen solution in 10 mM sodium acetate (pH 4.0) was injected. Residual activated carboxylic groups were blocked with 150 μl of 1 M ethanolamine. A control flow cell was activated and blocked in the same manner without collagen injection. To measure the association of inactive mutants with collagens, a purified inactive mutant enzyme (130–652 nM diluted in 10 mM Tris–HCl (pH 8.0) containing 10 mM CaCl2) was applied to the immobilized collagen at a flow rate of 20 μl/min at 25 °C. After binding of inactive mutants to collagens, dissociation was observed at the same flow rate for 10 min.

Y. Uesugi et al. / Biochimica et Biophysica Acta 1814 (2011) 1295–1304

1297

Blunt II-TOPO; the resultant plasmids were confirmed by DNA sequencing. The plasmids representing SGT-Q72R and SGT-L71Y-Q72R were digested with NdeI and KpnI. The plasmid representing the partial sprT was digested with KpnI and HindIII. The fragments were ligated into the NdeI-HindIII gap of pTONA5a to construct the expression vector. The expression and purification of the mutants were done similarly to SGT, as described above; their purities were confirmed using SDS-PAGE.

2.6. Construction of inactive chimeras We constructed inactive chimeras between SOT and SGT to investigate which domain is related to the interaction with collagens. A chimeric gene library between SOT and SGT was constructed using RIBS in vivo DNA shuffling, as detailed previously [9]. Among them, we chose eight chimeras: A–H. To prepare the inactive chimeras A–E, the genes encoding chimeras A–E were digested using NdeI and Aor51HI and ligated into the NdeI-Aor51HI gap of pTONA5a(SGT-S172A). To prepare inactive chimeras F–H, the mutagenic gene was amplified using PCR with a combination of a forward primer (5′-GCGTCGACACCTGTCAGGGCGAC(T → G, Ser172 → Ala)CCGG-3′, corresponding to nt 608– 635 from sot) and a reverse primer (5′-TGCCGGTACGAAGCTTCAGAGCGTGCG-3′, corresponding to the HindIII site of sprT) using each chimeric gene as the template. The amplified DNA fragment was then cloned, sequenced, and digested with SalI and HindIII. The plasmid pCRBlunt II-TOPO (SOT) was digested with NdeI and SalI. Fragments of the sot gene and each mutagenic gene were ligated into the NdeI–HindIII gap of pTONA5a to construct the expression vector. Next, to identify the amino acid residues related to the interaction with collagens, we further constructed the inactive chimera B and C mutants. Mutants B-1–B-4 were constructed using PCR amplification, as detailed previously [9]. The genes representing B-1 and B-4 were digested with NdeI and Aor51HI. Each fragment was ligated into the NdeI–Aor51HI gap of pTONA5a(SGT-S172A) to construct the expression vector. The expression and purification of inactive chimeras were done similarly to SOT or SGT as described above; their purities were confirmed using SDS-PAGE.

2.8. Assay for enzyme activity To estimate the substrate specificity of the enzymes, hydrolytic activities were determined using fluorogenic bovine skin DQ-collagen type I and human placenta DQ-collagen type IV as described in our previous paper [3]. The assay was carried out as follows: 10 μl of the enzyme solution and 10 μl of 1 mg/ml DQ-collagen were added to 100 μl of 50 mM Tris–HCl (pH 8.0) containing 10 mM CaCl2 in a well of a micro-titer plate for fluorescence and incubated at 37 °C. After preincubation at 37 °C for 5 min, the reaction was started by adding the enzyme solution; it was subsequently monitored to assess the increase in fluorescence intensity at λex 485 nm and λem 535 nm using a multilabel counter (ARVO 1420; PerkinElmer Inc.). The reaction velocity was estimated from the standard curve shown with data obtained using fluorescein isothiocyanate (FITC). One unit of activity was defined as the amount of the enzyme necessary to release 1 nmol FITC min −1 under these assay conditions.

2.9. Collagenolytic activities using native collagens 2.7. Construction of SGT mutants

Collagenolytic activities of the enzymes were determined using native insoluble type I collagen from bovine Achilles tendon and type IV collagen from human placenta based on the procedure described in an earlier report [23]. The reaction mixture contained 2 mg of collagen, 0.4 ml of 50 mM Tris–HCl (pH 8.0) containing 10 mM CaCl2, and 0.1 ml of enzyme solution. After preincubation at 37 °C for 5 min, the reaction was started by adding the enzyme solution and incubating at 37 °C with shaking (1000 rpm) for 30–60 min. The control mixture did not contain the enzyme. The reaction was stopped by adding 1 μl of 0.2 M HCl. The initial rate of increase in free amino groups was measured using the ninhydrin method. After centrifugation at 4 °C and 10,000g for 10 min, 0.1 ml of supernatant was mixed with 0.4 ml of Ninhydrin Colour Reagent Solution (Nacalai Tesque Inc.) and heated to 97 °C for 10 min followed by chilling of the mixture on ice. Then 1 ml of 2-propanol was added to the mixture; the increased absorbance of the supernatant was determined at 570 nm. Clostridium histolyticum collagenase type I was also estimated as a reference enzyme. One collagen digestion unit liberates peptides from collagen by collagenase type I equivalent in ninhydrin color to 1.0 μmol of leucine in 5 h at pH 7.4 at 37 °C in the presence of calcium ions.

We constructed mutants in which the identified key residues 71 and 72 were substituted in wild-type SGT and SOT to investigate the effects of the identified residues on substrate recognition. To prepare mutants SGT-Q72R and SGT-L71Y-Q72R, the following two mutagenic anti-sense primers, where the KpnI site (underlined) was substituted with a silent mutation, were synthesized: 5′-CGGT(G→ A)CCGTTGTAGCCGGGGGC (CT → TC, Gln72→ Arg)GGAGGACCTTG-3′ (corresponding to nt 315– 349 from sprT) and 5′-CGGT(G→ A)CCGTTGTAGCCGGGGGC(CT → TC, Gln72 → Arg)GG(AG → TA, Leu71→ Tyr)GACCTTG-3′ (corresponding to nt 315–349 from sprT). The target mutation was introduced with primer sets of 5′-CAACATATGAAGCACTTCCTGCGTGC-3′ (a sense primer, corresponding to the NdeI site of sprT) and each of the mutagenic primers using KOD-Plus. The partial sprT gene was amplified using PCR with a combination of a forward primer (5′-CCGGCTACAACGG(C → T) ACCGGCAA-3′ for silent mutation of the KpnI site (underlined), corresponding to nt 332–353 from sprT) and a reverse primer (5′TGCCGGTACGAAGCTTCAGAGCGTGCG-3′, corresponding to the HindIII site of sprT). The amplified DNA fragments were cloned into the pCR-

Amino acid sequence

Enzyme SOT SOT-S172A SOT-Y71L-S172A SGT SGT-S172A SGT-L71Y-S172A

58

Q Q Q Q Q Q

S S S S S S

S S S S S S

S S S S S S

A A A A A A

I I I V V V

K K K K K K

V V V V V V

R R R R R R

S S S S S S

T T T T T T

K K K K K K

I I I V V V

Y Y L L L Y 71

R R R Q Q Q

72

S A A S A A

172

172

Fig. 1. Construction of inactive SOT, SGT, and their mutants. (A) Amino acid sequences of active and inactive SOT, SGT, and their mutants. The gray box shows the catalytic Ser residue mutated to Ala. The identified residue related to topological specificity is shown in the black box.

1298

Y. Uesugi et al. / Biochimica et Biophysica Acta 1814 (2011) 1295–1304

A

B

2500

2500

SOT-S172A

SOT-Y71L-S172A 2000

652 nM 1500

Response (RU)

Response (RU)

2000

435 nM 291 nM

1000

191 nM 130 nM

500

1500

652 nM 435 nM

1000

291 nM 191 nM 130 nM

500

0

0 0

200

400

600

800

1000

0

200

Time (sec)

C

D

2500

Response (RU)

Response (RU)

1500

652 nM 435 nM 291 nM 191 nM 130 nM

1000

1500

652 nM

1000

435 nM 291 nM 191 nM 130 nM

500

0 0

200

400

600

800

1000

0

200

Time (sec)

F

2500

800

1000

2500

2000

Response (RU)

435 nM 1500

600

SOT-Y71L-S172A

652 nM

2000

400

Time (sec)

SOT-S172A Response (RU)

1000

SGT-L71Y-S172A

0

291 nM 191 nM

1000

130 nM

1500

652 nM 435 nM

1000

291 nM 191 nM 130 nM

500

500

0

0 0

200

400

600

800

1000

0

200

Time (sec)

400

600

800

1000

Time (sec)

H

2500

2500

SGT-S172A

SGT-L71Y-S172A

2000

2000

Response (RU)

Response (RU)

800

2000

500

G

600

2500

SGT-S172A 2000

E

400

Time (sec)

1500

652 nM

1000

435 nM 291 nM 191 nM 130 nM M

500

1500

652 nM 435 nM

1000

291 nM 191 nM 130 nM

500

0

0 0

200

400

600

Time (sec)

800

1000

0

200

400

600

Time (sec)

800

1000

Y. Uesugi et al. / Biochimica et Biophysica Acta 1814 (2011) 1295–1304

A

B 2000

Collagen type I

1500

Response (RU)

Response (RU)

2000

pH 8

N.S.

1000

500

1000

500

0 SOT SOT-Y71L SGT SGT-L71Y -S172A -S172A -S172A -S172A

C

SOT SOT-Y71L SGT SGT-L71Y -S172A -S172A -S172A -S172A

D 2000

Collagen type I Response (RU)

2000

Response (RU)

Collagen type IV

1500

0

pH 4

1299

1500

1000 N.S.

Collagen type IV

1500

1000 N.S.

500

500

0

0 SOT SOT-Y71L SGT SGT-L71Y -S172A -S172A -S172A -S172A

SOT SOT-Y71L SGT SGT-L71Y -S172A -S172A -S172A -S172A

Fig. 3. Effects of pH on interaction of inactive mutants with native collagens. The maximal responses of SOT-S172A, SGT-S172A, SGT-L71Y-S172A, and SOT-Y172L-S172A were obtained from their specific associations when they passed over type I and type IV collagens at 435 nM at a flow rate of 20 μl/min under pH 8 or 4. Each value represents the mean ± SD from three independent experiments. The significant difference between SOT-Y172L-S172A and SOT-S172A and between SGT-L71Y-S172A and SGT-S172A is indicated with ***P b 0.0001, **P b 0.001, *P b 0.01, and N.S.: P N 0.05 by t test.

2.10. Assay of hydrolytic activity using a short peptidic substrate

3. Results

For the estimation of hydrolytic activity toward a short peptidic substrate, Z-Gly-Pro-Arg-MCA was used because it is a collagen mimic substrate. The assay was performed as follows: 20 μl of the enzyme solution was added to 180 μl of 0.2 mM Z-Gly-Pro-Arg-MCA in 50 mM Tris–HCl (pH 8.0) containing 10 mM CaCl2 in a micro-titer plate well for fluorescence and incubated at 37 °C. After preincubation at 37 °C for 5 min, the reaction was started by adding the enzyme solution; it was subsequently monitored to assess the increase in fluorescence intensity at λex 390 nm and λem 460 nm using a grating microplate reader (SH8000Lab; CORONA electric Co., Ltd). The reaction velocity was estimated from the standard curve plotted using 7-amino-4-methylcoumarin (AMC). In the kinetic assays, the reactions were carried out in a mixture consisting of Z-Gly-Pro-Arg-MCA at a final concentrations of 0.03–0.5 mM in 50 mM Tris–HCl (pH 8.0) containing 10 mM CaCl2 and the purified enzymes under the assay conditions described above.

3.1. Preparation of inactive mutants

2.11. Statistical analysis All statistical evaluations were performed using an unpaired Student's t test. All data are presented as mean ± standard deviation (SD) of at least three determinations.

Previously, we found that Tyr71 of SOT (corresponding to residue 89 of α-chymotrypsin numbering) is a key amino acid residue conferring topological specificity [9]. In this study, to evaluate the effect of the identified key residue on interaction with structural protein substrate by SPR analysis, we constructed inactive mutants of SOT and SGT, in which the catalytic Ser172 (corresponding to residue 195 of α-chymotrypsin numbering) was substituted with Ala (Fig. 1A). Then, we expressed the resultant genes and purified recombinants. All purified mutants showed mostly a single band with equal molecular weight (approximately 23 kDa) to those of wild-type SOT and SGT on SDS-PAGE (SI Fig. 10A). All mutants in which Ser172 was substituted with Ala lost hydrolysis activities toward collagen. By the analysis of N-terminal amino acid sequences, the sequences of active and inactive SOT were determined to be VVGGTRA. Thus, these active and inactive enzymes were matured by truncation of a signal peptide and a pro peptide. Their CD spectra were measured to confirm the folding of the inactive mutants of SOT and SGT. SI Fig. 10B and C presents spectra showing that inactive mutants folded with a secondary structure similarly to their active forms. In addition,

Fig. 2. Quantitative SPR analysis of interaction of inactive mutants and native collagens. Sensorgrams at different concentrations of SOT-S172A (A, E), SGT-S172A (C, G), SOT-Y71LS172A (B, F), and SGT-L71Y-S172A (D, H) are shown. As substrate, native type I (A–D) or type IV (E–H) collagens were immobilized on a CM5 sensor chip. Sensorgrams were obtained when inactive mutants were passed over collagen at 652, 435, 291, 191, and 130 nM, respectively (from top to bottom), with 10 mM Tris–HCl (pH 8.0) containing 10 mM CaCl2 at a flow rate of 20 μl/min for 5 min at 25 °C, followed by a buffer at the same flow rate for 10 min.

1300

Y. Uesugi et al. / Biochimica et Biophysica Acta 1814 (2011) 1295–1304

tryptophan fluorescence emissions of inactive mutants were also similar to those of their active mutants (data not shown). These results suggest that we prepared inactive mutants successfully.

A

Ser172

inactive SGT

Ala

223 aa

Chimera A

3.2. Interaction of inactive mutants with collagens

Chimera C Chimera D Chimera E Chimera F Chimera G Chimera H SOT

223 aa

0

50

100

150

200 223 aa

B Response (RU)

2000

Type I

1500

1000

500

0 SGT

A

B

C

D

E

F

G

H

SOT

C

D

E

F

G

H

SOT

C 2000

Response (RU)

We analyzed the binding profiles of SOT-S172A, SGT-S172A, SOTY71L-S172A, and SGT-L71Y-S172A for native type I collagen from bovine Achilles tendon and type IV collagen from human placenta by SPR analysis. Sensorgrams showed real-time biomolecular interaction. Overlaid sensorgrams were obtained when SOT-S172A, SGT-S172A, SOT-Y71L-S172A, and SGT-L71Y-S172A were passed at different concentrations over immobilized type I (Fig. 2A–D) and type IV (Fig. 2E–H) collagens. These results show that SOT-S172A exhibited much higher binding ability for both collagens than SGT-S172A at all concentrations. Interactions of SOT-Y71L-S172A with type I and type IV collagens decreased significantly in comparison to those of SOT-S172A. In contrast, the interaction of SGT-L71Y-S172A for type IV collagen was somewhat higher than that of SGT-S172A. Interestingly, in the cases of SOT-S172A and SGT-L71Y-S172A, the increase rates of interaction toward type IV collagen with enzyme concentrations were greater than those toward type I collagen. In contrast, no observation of these trends existed in cases of SGT-S172A and SOT-Y71L-S172A. Collagen substrates probably have many nonequivalent binding sites for SOT and SGT mutants. It is possible that collagens are unfavorable substrates for the ideal SPR analysis which is obtained kinetic constant KD. Therefore, maximal responses of these inactive mutants toward collagens were compared. Fig. 3A and B shows maximal responses at 435 nM of these inactive mutants toward collagens. It is noteworthy that SGT-L71YS172A exhibited a significantly higher interaction than SGT-S172A for type IV collagen (t test; P b 0.01), although no significant difference existed between them for type I collagen (t test; P N 0.05). On the other hand, the difference between SOT-Y71L-S172A and SOT-S172A for type IV collagen was significant and greater than that for type I collagen (t test; P b 0.0001 for type IV collagen, P b 0.001 for type I collagen). We further investigated the effect of pH conditions on the interactions of SOT-S172A, SGT-S172A, SOT-Y71L-S172A, and SGTL71Y-S172A toward collagens. At each pH condition, SOT-S172A showed a significant interaction with both collagens when compared to SGT-S172A (t test; P b 0.01–0.0001). At the acidic pH condition (pH 4), the interactions of all inactive mutants decreased more than those at the alkaline pH condition (pH 8) (Fig. 3). The pH conditions affected both the degree and order of interactions among inactive mutants. These results showed that the substitution Leu71 with Tyr induced difference of binding abilities toward type I collagen and type IV collagen. However, SGT-L71Y-S172A has less binding ability toward collagens than SOT-S172A. There is remarkable difference between SOT-S172A and SGT-S172A in the binding ability toward collagens. Therefore, we infer that other residues contribute to collagen binding, although residue 71 plays a role in recognizing the structural protein substrate.

Chimera B

Type IV

1500

1000

500

0 SGT

A

B

Inactive mutant Fig. 4. Interaction of inactive chimeras A–H with native collagens. (A) Primary structures of eight inactive chimeras, parental SGT and SOT are illustrated schematically. The arrowhead indicates Ser172 of the catalytic triad was mutated to Ala. The maximal responses of inactive chimeras, parental SGT and SOT were measured for their specific associations with native type I (B) and type IV (C) collagens at pH 8.0. Each enzyme (435 nM) was injected at a flow rate of 20 μl/min. Data are expressed as mean± SD of three independent experiments.

3.3. Comparison of interaction for chimeras with collagens

3.4. Identification of amino acid residue(s) related to collagen binding

To investigate which region is involved in collagen binding, we first constructed chimeras of SOT with SGT by RIBS in vivo DNA shuffling [9] (Fig. 4A). Next, we constructed inactive chimeras, in which the catalytic Ser172 was substituted with Ala, for SPR analysis. The interaction of purified inactive chimeras with native type I and type IV collagens was analyzed (Fig. 4B and C). Inactive chimeras A and B had much lower interaction than other inactive chimeras and SOT. We found a considerable difference between inactive chimeras B and C (Fig. 4B and C). These results suggest that the region between inactive chimeras B and C (corresponding to residues 52–72 of SOT) is related to the collagen binding. Therefore, we further examined the correlation between this region and collagen binding.

As shown in Fig. 5A, the five amino acid residues of the inactive chimera B were different from those of chimera C. Therefore, we constructed four inactive chimera B mutants (B-1–B-4; their primary sequence is presented in Fig. 5A) and evaluated their interaction for collagens. For both collagen types, the interactions of inactive chimera B mutants were considerably lower than those of inactive chimera C (Fig. 5B and C). Fig. 5A portrays that inactive chimeras B-4 and C differed in only one amino acid residue: residue 72 (corresponding to residue 90 of α-chymotrypsin numbering). From these results, we inferred that residue 72 is a key amino acid residue conferring collagen binding. Furthermore, we attempted to confirm the contributions of residues 71 and 72 to collagen binding using inactive SGT

Y. Uesugi et al. / Biochimica et Biophysica Acta 1814 (2011) 1295–1304

1301

A Amino acid sequence B B-1 B-2 B-3 B-4 C

B

52

G A A A A A

G G G G G G

V V V V V V

V V V V V V

D D D D D D

L L L L L L

Q Q Q Q Q Q

S S S S S S

S S S S S S

C

2000

S S S S S S

A A A A A A

K K K K K K

V V V V V V

R R R R R R

S S S S S S

T T T T T T

K K K K K K

V V V I I I

L L L L Y Y

Q Q Q Q Q R

72

2000

Collagen type I

Collagen type IV

1500

Response (RU)

Response (RU)

V V I I I I

1000

500

1500

1000

500

0

0 B

B-1

B-2

B-3

B-4

C

B

B-1

B-2

B-3

B-4

C

Inactive mutant

Inactive mutant

Fig. 5. Identification of an amino acid residue related to the interaction with collagens. Amino acid sequences of inactive chimeras B and C mutants (A), and their interaction with native type I (B) and type IV (C) collagens are shown. The maximal responses of inactive chimeras (435 nM) were measured for their specific associations with collagens at pH 8.0. Each value represents the mean ± SD from three independent experiments.

mutant, in which Leu71 and Gln72 were substituted respectively with Tyr and Arg. It was disappointing that the folding of the inactive mutant differed from that of its active mutant (data not shown). For that reason, we were unable to analyze it further. 3.5. Effect of mutations on the hydrolytic activity We prepared a single and double mutant of SGT and SOT to investigate the effect of the residues Tyr71 and Arg72 of SOT on the activity. We used fluorogenic bovine skin collagen type I and human placenta collagen type IV as substrates; we then compared hydrolytic activities of the mutants with those of wild-type SOT and SGT (Fig. 6). Our prior study showed that the results of hydrolysis activities of SOT and SGT toward fluorescein-labeled collagens agreed well with those toward native collagens [3]. Fig. 6A shows that SGT mutants SGTL71Y, SGT-Q72R, and SGT-L71Y-Q72R had slightly lower activities toward type I collagen than wild-type SGT. In contrast, the hydrolytic activities of SGT mutants toward type IV collagen were higher than that of SGT (Fig. 6B). In particular, the double mutant of SGT (SGTL71Y-Q72R) showed 5.2-fold higher specific activity toward type IV collagen than wild-type SGT. Furthermore, it is noteworthy that mutant SOT-Y71L shows 3.5-fold lower specific activity toward type IV collagen than wild-type SOT, although SOT-Y71L displayed high activity toward type I collagen similar to SOT. From these specific activities, the ratio of the hydrolytic activity toward type IV collagen to that toward type I collagen (collagen IV/I) was calculated (Fig. 6C). The ratios of SGT-L71Y and SGT-Q72R were more than twice as high as that of SGT. Interestingly, SGT-L71Y-Q72R showed a substantially higher ratio than SGT and SOT. Moreover, we evaluated the effect of the mutation on the specificities for native type I collagen substrates from bovine Achilles tendon and type IV collagen from human placenta (Fig. 7). The activities of SGT-L71Y, SOT-Y71L, SGT-L71Y-Q72R, wild-type SOT, and SGT were compared. The effect of the mutation on the specificities appeared more clearly for native collagen substrates. The collageno-

lytic activities of SGT-L71Y and SGT-L71Y-Q72R for type IV collagen were 3.3-fold and 4.6-fold, respectively, higher than that of SGT (Fig. 7B), although their activities toward type I collagen were somewhat lower than that of SGT (Fig. 7A). In contrast, SOT-Y71L and SOT hydrolyzed type I collagen similarly, but the activity of the former toward type IV collagen was 2.2-fold lower than that of the latter. As shown in Fig. 7C, collagen IV/I of SGT-L71Y and SGT-L71Y-Q72R were 4.8-fold and 6.3-fold, respectively, higher than that of SGT, whereas it was two-fold lower for SOT-Y71L compared to SOT. Therefore, we conclude that residue 72 confers topological specificity synergistically with residue 71. The ability of the mutation to hydrolyze the collagen mimic substrate was determined using Z-Gly-Pro-Arg-MCA, which is frequently used as a substrate in studies of collagenolytic cysteine proteinase, cathepsin K [24,25]. Table 1 shows that specific activities were 1.4-fold and 1.7-fold higher in SGT-L71Y and SGT-L71Y-Q72R, respectively, compared to that of SGT, and it was 2.6-fold lower in SOT-Y71L compared to SOT. For this short peptidic substrate, substitution of residues 71 and 72 has little effect, unlike the response seen with the structural protein substrates. The kcat values of SGT-L71Y (51.3 s −1) and SGT-L71Y-Q72R (57.3 s −1) were 1.6-fold and 1.7-fold higher than that of SGT (33.0 s −1). Although Km values of SGT-L71Y and SGT were equivalent, it was lower for SGT-L71Y-Q72R compared to SGT. These results suggest that residues 71 and 72 also affect significantly catalytic efficiency. 4. Discussion We recently found that Tyr71 of SOT (corresponding to residue 89 of α-chymotrypsin numbering) is the key amino acid residue conferring topological specificity [9]. In the present study, we first estimated the effect of this residue on interaction with structural protein substrate using SPR analysis to investigate the role of this residue in substrate recognition in greater detail. To date, we have

1302

Y. Uesugi et al. / Biochimica et Biophysica Acta 1814 (2011) 1295–1304

200

A

150

100

50

0

5000

Collagen type I

Specific activity (U/mg)

Specific activity (U/mg)

A

SGT

SGTL71Y

SOTY71L

Collagen type I 4000 3000 2000 1000

SGT- SGT-L71Y SOT Q72R -Q72R

0

SGT

B B

Collagen type IV

80

Specific activity (U/mg)

Specific activity (U/mg)

100

60 40 20 0

SGT

SGTL71Y

SOTY71L

SGT- SGT-L71Y SOT Q72R -Q72R

SOTY71L

SGT-L71Y -Q72R

SOT

SOTY71L

SGT-L71Y -Q72R

SOT

20000 15000 10000 5000

C

SGT

SGTL71Y

8 7

0.5

Type IV / I

6

0.4 0.3 0.2 0.1 0

SOT

Collagen type IV

Collagen IV/I

Collagen IV/ I

Type IV / I

0.6

SGT-L71Y -Q72R

25000

0.8 0.7

SOTY71L

30000

0

C

SGTL71Y

5 4 3 2

SGT

SGTL71Y

SOTY71L

SGT- SGT-L71Y SOT Q72R -Q72R

Fig. 6. Effects of mutations on substrate specificity. The reaction was performed using bovine skin DQ-collagen type I (A) and human placenta DQ-collagen type IV (B) in 50 mM Tris–HCl (pH 8.0) containing 10 mM CaCl2 at 37 °C, as described in Materials and methods. The enzyme activity was measured by monitoring the fluorescein (FITC) release (ex: 395 nm; em: 415 nm). Data are expressed as mean ± SD of three independent experiments. (C) The ratio of hydrolytic activity toward type IV collagen to that toward type I collagen.

demonstrated that the N-terminal HKD motif of Streptomyces phospholipase D (PLD) contains the nucleophile, and clarified roles of the key residues in PLD on substrate recognition using SPR analysis [26,27]. SPR analysis showed that residue 71 certainly affected not only topological specificity but also interaction with collagens. However, the effect was insufficient to gain binding ability as great as that of SOT. Consequently, we inferred that other residues contribute to collagen binding. Moreover, inactive mutants were more bound to type IV collagen at pH 8.0 than at pH 4.0, although the binding abilities of inactive mutants for type I collagen were little affected by pH (Fig. 3). Our previous report described that the basic surface-charged regions dominate the SOT surface [9]. These results suggest that type IV collagen has more exposed carboxyl groups, which contribute to interaction with inactive mutants than type I collagen, because the side chain carboxyl groups of Asp and Glu are mainly un-ionized under their pKa value (approximately pKa 4.7). Using the shuffling system, we also identified Arg72 (corresponding to residue 90 of α-chymotrypsin numbering) in the basic surface

1 0

SGT

SGTL71Y

Fig. 7. Hydrolytic activities of SOT, SGT, and their mutants toward native collagens. Collagenolytic activity was determined by the following method. After preincubation of 2 mg of insoluble type I collagen from bovine Achilles tendon (A) or type IV collagen from human placenta (B) in 50 mM Tris–HCl (pH 8.0) containing 10 mM CaCl2, enzyme was added and incubated at 37 °C for 30–60 min. Next, the reaction was terminated by adding 1 μl of 0.2 M HCl; the rate of increase in free amino groups was measured using the ninhydrin method. Clostridium histolyticum collagenase type I liberates peptides from collagen, and one collagen digestion unit is equivalent to ninhydrin color change to 1.0 μmol of leucine in 5 h at pH 7.4 at 37 °C in the presence of CaCl2. Data are expressed as mean ± SD of three independent experiments. (C) The ratio of hydrolytic activity toward type IV collagen to that toward type I collagen.

Table 1 Hydrolysis of a short peptidic substrate by SOT, SGT, and their mutants.

SGT SGT-L71Y SOT-Y71L SGT-L71Y-Q72R SOT a

Specific activity (μmol/min/mg)a

Km (μM)b

kcat (s−1)b

kcat/Km (μM−1 s−1)b

66.1 ± 4.7 90.3 ± 8.3 190.2 ± 15.0 110.2 ± 8.4 501.5 ± 44.4

17.7 ± 0.6 17.2 ± 5.7 17.5 ± 2.9 12.0 ± 1.7 6.1 ± 1.9

33.0 ± 4.1 51.3 ± 7.8 93.0 ± 17.1 57.3 ± 1.8 279.5 ± 12.1

1.9 3.2 5.4 4.8 49.7

Enzyme activity was determined based on the hydrolysis of 0.2 mM Z-Gly-Pro-Arg-MCA. The assay was carried out using 0.03–0.5 mM Z-Gly-Pro-Arg-MCA in 50 mM Tris–HCl (pH 8.0) containing 10 mM CaCl2 at 37 °C. Data are expressed as mean± SD of three independent experiments. b

Y. Uesugi et al. / Biochimica et Biophysica Acta 1814 (2011) 1295–1304

regions of SOT as an important residue for collagen binding (Figs. 4 and 5). By substituting Leu71 and Gln72 with Tyr and Arg, respectively, SGT mutant showed a marked change in topological specificity and high hydrolytic activity toward type IV collagen comparable to SOT (Figs. 6 and 7). Our previous study found that only residue 71 is crucial for substrate recognition by hydrolysis assay [9]. In this study, we found another key residue 72 for substrate recognition because of the SPR analysis using inactive mutants, in which the catalytic Ser was substituted with Ala, for determining the interaction for the enzyme– substrate complex before the tetrahedral intermediate via Ser. In our recent study, we overlooked residue 72 as a key on topological specificity using active chimeras and different types of collagens [9]. We assume that the key residue 72 was obscured from our past search behind formation of the tetrahedral intermediate via the catalytic Ser. This supposition also agrees with results showing that the binding ability of inactive SOT was about two-fold higher than that of inactive SGT (Fig. 2), although the hydrolytic activity of SOT toward type IV collagen was about six-fold higher than that of SGT (Fig. 7B). In other studies, detailed information of the collagen-binding domain (CBD) in Clostridium histolyticum collagenase has been analyzed using SPR analysis [16,17]Clostridium histolyticum collagenases belong to metalloproteases and are the most well-known bacterial collagenases. They are grouped into class I and class II (ColG and ColH) [28,29]. In fact, ColG possesses tandem CBDs at C-terminus, whereas ColH contains only one. The CBD consists of about 110 amino acids and is revealed to possess a β-sheet sandwich fold with the N-terminal linker adopting an α-helix [17,30]. Perona et al. showed that Tyr994 in this domain is the critical residue for interaction with collagen interaction; the hydroxyl group of this residue is likely to be hydrogen binding to the main-chain atoms for a protein-collagen complex [31]. Our result showed that Tyr71 and Arg72 of SOT contributed to collagen binding. The hydroxyl group of Tyr71 is also likely to form hydrogen bonds with the main-chain atoms as similar to Tyr994 of CBD. Additionally, we speculate that a guanidino group of Arg72 forms an ion bridge with a carboxyl group of Asp and Glu of collagens in a sequence-independent manner. These considerations agree with the feature of SOT, which can degrade both helical and non-helical collagens. Chymotrypsin-like serine proteases possess an identical fold consisting of two β-barrel domains with similar topology. The catalytic triad consists of Ser, His, and Asp located at the interface of the two domains [32]. The specificity at the S1 site has been explained using the S1 pocket structure: it is formed by three β-sheets (residues 189–192, 214–216, and 224–228, corresponding to chymotrypsin numbering) connected by two surface loops and the disulfide bond Cys191 and Cys220 [32] in the C-terminal β-barrel domain. Amino acid side chains at positions 190 and 228 extend into the base of the pocket and modulate the specificity profile, whereas residues 216 and 226 are additional primary determinants. At the base of the S1 pocket, Asp189 (corresponding to Asp166 in this study) is a primary determinant of Arg and Lys specificity of trypsin. Additionally, other report of thrombin described that Ser214, locating adjacent to active site and forming part of the primary specificity pocket, plays a role in promoting the formation of the enzyme–substrate complex [33]. Based on the crystal structure of SGT [12], our identified key residues 71 and 72 are present in the β-sheet of the N-terminal β-barrel domain (Fig. 8A). An interesting observation was the location of the residues 71 and 72; these residues were located at just a symmetric position to the S1 site described above across the catalytic triad. The C-terminal β-barrel domain of serine proteases, especially residues 189–220, contains most of the structural determinants important for direct substrate recognition at the S1–S3 sites [34] in addition to the Na + binding site of allosteric proteases [35,36]. Residues Asp189, Glu217, Asp222, and Tyr225 of thrombin are in the allosteric core controlling Na+ binding and mutation of these residues with Ala caused more 30-fold decrease in affinity to Na+[36]. This

1303

A

D166

D82

Catalytic triad

W83 S172

H37 R72 Y71

B D82 S172

H37 W83

R72

Y71

Fig. 8. The amino acid residues conferring topological specificities and interaction with structural protein substrate in the three-dimensional structure. (A) The overall structure of SOT is portrayed using the Swiss-pdb viewer based on the crystal structure of SGT. The key residues, Tyr71 and Arg72 of SOT, are shown respectively in red and pink. Asp82, His37, and Ser172 of the catalytic triad are presented as purple, yellow green, and blue, respectively. (B) The local environment around the identified key residues (Tyr71 and Arg72 of SOT) is shown using the Swiss-pdb viewer.

domain has been regarded as accounting fully for the functional diversity of serine proteases [34]. Therefore, it is noteworthy that two key residues conferring topological specificity and interaction with structural protein substrate exist in the N-terminal β-barrel domain. Fig. 8A presents that residue 72 is located at the entrance of the catalytic site in the domain-domain interface. It is reasonable to consider that residue 72 plays a role in substrate recognition from a geometrical viewpoint. On the other hand, residue 71 is more distant from the catalytic triad, and is exposed to a solvent. In a previous study, we showed that residue 71 is crucial not only for topological specificity but also for enzyme conformation and folding [9]. Because residue 71 is located adjacent to Trp83 (corresponding to residue 103 of α-chymotrypsin numbering) (approximately 3 Å), residue 71 is expected to interact with Trp83, with a subsequent change in the local environment around the catalytic Asp82 (corresponding to the residue 102 of α-chymotrypsin numbering). Ishida et al. reported that Asp102 of trypsin plays two important roles in the catalytic process: one is to stabilize the protonated His57, or ionic intermediate, formed during the acylation, and the other is to fix the configuration around the

1304

Y. Uesugi et al. / Biochimica et Biophysica Acta 1814 (2011) 1295–1304

active site, which is favorable to promote the catalytic process [37]. Additionally, the hydrolytic activity of SOT toward type I collagen resembled that of SGT despite differences in their conformation [9]. From these results, we infer that residue 71 probably plays roles in recognizing structural protein substrate and as a trigger of induced fitting when the enzyme–substrate complex is formed in the first step of the catalytic reaction. Consequently, the local environment around the active site presumably changes for attachment to the structural protein substrate and the side group of Arg72 of SOT is oriented appropriately for the acidic surface of collagens. This structural modification might engender changes in binding ability for substrates, topological specificity, and catalytic efficiency. In fact, the double mutant of SGT, in which Leu71 and Gln72 were substituted respectively with Tyr and Arg, showed a much change in topological specificity (Figs. 6 and 7). Its collagen IV/I was more than 6-fold than that of SGT and higher than that of wild-type SOT. Moreover, catalytic efficiency of SOT and SGT on hydrolysis of a short peptide when carrying a mutation at these residues (Table 1). The kcat/Km value of SGT-L71Y-Q72R was 2.5-fold higher than that of SGT. In particular, the value of SOT-Y71L was lower than that of SOT by 1 order of magnitude. Comparison of this study to our recent results implies that residues 71 and 72 play different roles in substrate recognition. Residue 71 recognizes structural protein substrate and affects the local environment of the catalytic site via Trp83. It also affects hydrolytic activity, collagen binding, enzyme conformation, and folding, whereas residue 72 acts on binding to collagens via an ion bridge. Furthermore, the neighboring residues work synergistically in the catalytic reaction. In conclusion, we identified the key residues 71 and 72 conferring topological specificity and interaction with structural protein substrate. Results suggest that these residues synergistically play an important role in substrate recognition. These findings promote understanding the substrate recognition mechanism, and contribute to development of industrial and medical applications in the serine protease world. Moreover, the key residues Leu71 and Gln72 of SGT (corresponding to Tyr71 and Arg72 of SOT) are highly conserved in homological trypsins and trypsinogens from Streptomyces except SOT. Therefore, SOT shows higher collagen degradation than SGT. Furthermore, SOT has a compact structure with a catalytic triad between two domains containing functional sites for substrate specificity at the S1– S3 sites, topological specificity, and interaction for structural protein substrate. Therefore, SOT can become an ideal scaffold for tailor-made biocatalyst possessing a range of useful properties. Supplementary materials related to this article can be found online at doi:10.1016/j.bbapap.2011.06.015. Acknowledgements This research was financially supported by Grant-in Aid for Scientific Research (21750183). References [1] N.D. Rawlings, A.J. Barrett, MEROPS: the peptidase database, Nucleic Acids Res. 28 (2000) 323–325. [2] M.M. Krem, E. Di Cera, Molecular markers of serine protease evolution, EMBO J. 20 (2001) 3036–3045. [3] Y. Uesugi, J. Arima, H. Usuki, M. Iwabuchi, T. Hatanaka, Two bacterial collagenolytic serine proteases have different topological specificities, Biochim. Biophys. Acta 1784 (2008) 716–726. [4] Y. Itoi, M. Horinaka, Y. Tsujimoto, H. Matsui, K. Watanabe, Characteristic features in the structure and collagen-binding ability of a thermophilic collagenolytic protease from the thermophile Geobacillus collagenovorans MO-1, J. Bacteriol. 188 (2006) 6572–6579. [5] P. Bressollier, F. Letourneau, M. Urdaci, B. Verneuil, Purification and characterization of a keratinolytic serine proteinase from Streptomyces albidoflavus, Appl. Environ. Microbiol. 65 (1999) 2570–2576. [6] S. Sugimoto, T. Fujii, T. Morimiya, O. Johdo, T. Nakamura, The fibrinolytic activity of a novel protease derived from a tempeh producing fungus, Fusarium sp. BLB, Biosci. Biotechnol. Biochem. 71 (2007) 2184–2189.

[7] J. He, S. Chen, J. Gu, Identification and characterization of Harobin, a novel fibrino (geno)lytic serine protease from a sea snake (Lapemis hardwickii), FEBS Lett. 581 (2007) 2965–2973. [8] Z. Hui, H. Doi, H. Kanouchi, Y. Matsuura, S. Mohri, Y. Nonomura, T. Oka, Alkaline serine protease produced by Streptomyces sp. degrades PrP(Sc), Biochem. Biophys. Res. Commun. 321 (2004) 45–50. [9] Y. Uesugi, H. Usuki, M. Iwabuchi, T. Hatanaka, The role of Tyr71 in Streptomyces trypsin on the recognition mechanism of structural protein substrates, FEBS J. 276 (2009) 5634–5646. [10] R.W. Olafson, L. Jurasek, M.R. Carpenter, L.B. Smillie, Amino acid sequence of Streptomyces griseus trypsin. Cyanogen bromide fragments and complete sequence, Biochemistry 14 (1975) 1168–1177. [11] J.C. Kim, S.H. Cha, S.T. Jeong, S.K. Oh, S.M. Byun, Molecular cloning and nucleotide sequence of Streptomyces griseus trypsin gene, Biochem. Biophys. Res. Commun. 181 (1991) 707–713. [12] R.J. Read, M.N. James, Refined crystal structure of Streptomyces griseus trypsin at 1.7 Å resolution, J. Mol. Biol. 200 (1988) 523–551. [13] C.M. Kielty, M.E. Grant, Molecular, Genetic, and Medical Aspects, Connective Tissue and Its Heritable Disorders, Wiley-Liss, New York, 2002, pp. 159–221. [14] M. van der Rest, R. Garrone, Collagen family of proteins, FASEB J. 5 (1991) 2814–2823. [15] G.J. Wegner, A.W. Wark, H.J. Lee, E. Codner, T. Saeki, S. Fang, R.M. Corn, Real-time surface plasmon resonance imaging measurements for the multiplexed determination of protein adsorption/desorption kinetics and surface enzymatic reactions on peptide microarrays, Anal. Chem. 76 (2004) 5677–5684. [16] O. Matsushita, T. Koide, R. Kobayashi, K. Nagata, A. Okabe, Substrate recognition by the collagen-binding domain of Clostridium histolyticum class I collagenase, J. Biol. Chem. 276 (2001) 8761–8770. [17] J.J. Wilson, O. Matsushita, A. Okabe, J. Sakon, A bacterial collagen-binding domain with novel calcium-binding motif controls domain orientation, EMBO J. 22 (2003) 1743–1752. [18] L. Hedstrom, Serine protease mechanism and specificity, Chem. Rev. 102 (2002) 4501–4524. [19] K. Mori, T. Mukaihara, Y. Uesugi, M. Iwabuchi, T. Hatanaka, Repeat-lengthindependent broad-spectrum shuffling, a novel method of generating a random chimera library in vivo, Appl. Environ. Microbiol. 71 (2005) 754–760. [20] T. Hatanaka, H. Onaka, J. Arima, M. Uraji, Y. Uesugi, H. Usuki, Y. Nishimoto, M. Iwabuchi, pTONA5: a hyperexpression vector in Streptomycetes, Protein Expr. Purif. 62 (2008) 244–248. [21] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–685. [22] P.L. Coleman, H.G. Lathham Jr., E.N. Shaw, Some sensitive methods for the assay of trypsinlike enzymes, Methods Enzymol. 45 (1976) 12–26. [23] A. Wlodawer, M. Li, A. Gustchina, N. Tsuruoka, M. Ashida, H. Minakata, H. Oyama, K. Oda, T. Nishino, T. Nakayama, Crystallographic and biochemical investigations of kumamolisin-As, a serine-carboxyl peptidase with collagenase activity, J. Biol. Chem. 279 (2004) 21500–21510. [24] F. Lecaille, S. Chowdhury, E. Purisima, D. Bromme, G. Lalmanach, The S2 subsites of cathepsins K and L and their contribution to collagen degradation, Protein Sci. 16 (2007) 662–670. [25] F. Lecaille, Y. Choe, W. Brandt, Z. Li, C.S. Craik, D. Bromme, Selective inhibition of the collagenolytic activity of human cathepsin K by altering its S2 subsite specificity, Biochemistry 41 (2002) 8447–8454. [26] Y. Uesugi, K. Mori, J. Arima, M. Iwabuchi, T. Hatanaka, Recognition of phospholipids in Streptomyces phospholipase D, J. Biol. Chem. 280 (2005) 26143–26151. [27] Y. Uesugi, J. Arima, M. Iwabuchi, T. Hatanaka, Sensor of phospholipids in Streptomyces phospholipase D, FEBS J. 274 (2007) 2672–2681. [28] O. Matsushita, C.M. Jung, S. Katayama, J. Minami, Y. Takahashi, A. Okabe, Gene duplication and multiplicity of collagenases in Clostridium histolyticum, J. Bacteriol. 181 (1999) 923–933. [29] K. Yoshihara, O. Matsushita, J. Minami, A. Okabe, Cloning and nucleotide sequence analysis of the colH gene from Clostridium histolyticum encoding a collagenase and a gelatinase, J. Bacteriol. 176 (1994) 6489–6496. [30] O. Matsushita, C.M. Jung, J. Minami, S. Katayama, N. Nishi, A. Okabe, A study of the collagen-binding domain of a 116-kDa Clostridium histolyticum collagenase, J. Biol. Chem. 273 (1998) 3643–3648. [31] J.J. Perona, C.A. Tsu, C.S. Craik, R.J. Fletterick, Crystal structure of an ecotincollagenase complex suggests a model for recognition and cleavage of the collagen triple helix, Biochemistry 36 (1997) 5381–5392. [32] J.J. Perona, C.S. Craik, Evolutionary divergence of substrate specificity within the chymotrypsin-like serine protease fold, J. Biol. Chem. 272 (1997) 29987–29990. [33] M.M. Krem, S. Prasad, E. Di Cera, Ser(214) is crucial for substrate binding to serine proteases, J. Biol. Chem. 277 (2002) 40260–40264. [34] M.M. Krem, T. Rose, E. Di Cera, The C-terminal sequence encodes function in serine proteases, J. Biol. Chem. 274 (1999) 28063–28066. [35] Q.D. Dang, E. Di Cera, Residue 225 determines the Na(+)-induced allosteric regulation of catalytic activity in serine proteases, Proc. Natl. Acad. Sci. U. S. A. 93 (1996) 10653–10656. [36] E. Di Cera, Thrombin, Mol. Aspects Med. 29 (2008) 203–254. [37] T. Ishida, S. Kato, Role of Asp102 in the catalytic relay system of serine proteases: a theoretical study, J. Am. Chem. Soc. 126 (2004) 7111–7118.