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ATP-dependent leader peptide cleavage by NukT, a bifunctional ABC transporter, during lantibiotic biosynthesis
Journal of Bioscience and Bioengineering VOL. 108 No. 6, 460 – 464, 2009 www.elsevier.com/locate/jbiosc
ATP-dependent leader peptide cleavage by NukT, a bifunctional ABC transporter, during lantibiotic biosynthesis Mami Nishie,1 Kouki Shioya,1 Jun-ichi Nagao,2 Hiroyuki Jikuya,3 and Kenji Sonomoto1,3,⁎ Laboratory of Microbial Technology, Division of Microbial Science and Technology, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan 1 Department of Functional Bioscience, Section of Infection Biology, Fukuoka Dental College, 2-15-1 Tamura, Sawara-ku, Fukuoka 814-0193, Japan 2 and Bio-Architecture Center, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan 3 Received 30 April 2009; accepted 1 June 2009
NukT, a possible ABC transporter maturation and secretion (AMS) protein, may contribute to the cleavage of the leader peptide of NukA, which is the prepeptide of the lantibiotic nukacin ISK-1, and to nukacin ISK-1 transport. In this study, we reconstituted in vitro peptidase activity of the full-length NukT overexpressed in inside-out membrane vesicles of Staphylococcus carnosus TM300. We found that the presence of unusual amino acids in NukA is required for leader peptide cleavage. Furthermore, NukT peptidase activity was inhibited by phenylmethylsulfonyl fluoride, a serine/cysteine protease inhibitor; this finding strongly suggests that NukT, like other AMS proteins, is a cysteine protease. Interestingly, NukT peptidase activity depended on ATP hydrolysis. These results suggest that the N-terminal peptidase domain of NukT may cooperatively function with the C-terminal ATP-binding domain. This is the first in vitro study on lantibiotics that reports the processing mechanism of a full-length bifunctional ABC transporter. © 2009, The Society for Biotechnology, Japan. All rights reserved. [Key words: Lantibiotic; Post-translational modification; Peptidase; ABC transporter]
Lantibiotics, antimicrobial peptides produced by gram-positive bacteria, contain unusual (e.g., lanthionine) and dehydrated amino acids. Ribosomally synthesized lantibiotic prepeptides consist of an Nterminal leader peptide and a C-terminal propeptide that undergoes several post-translational modifications (1, 2). Type-A(I) lantibiotics (e.g., nisin) undergo the following modifications: LanB dehydrates serines and threonines, LanC forms thioether rings (involving coupling of dehydro residues to cysteins), LanT exports modified prepeptides, and LanP cleaves off N-terminal leader peptides from exported peptides (LanP is an extracellular serine protease) (1, 3, 4). Type-A(II) lantibiotics (e.g., lacticin 481) undergo LanM-mediated dehydration and cyclization, which results in the formation of unusual amino acids, LanTmediated cleavage of the N-terminal leader peptide (LanT is a bifunctional ABC transporter) and secretion of mature lantibiotics (5). LanT, specific to type-A(II) lantibiotics, is an ABC transporter maturation and secretion (AMS) protein; AMS proteins comprise an N-terminal putative peptidase domain, a transmembrane domain, and a C-terminal ATP-binding domain (6). It is predicted that the N- and Ctermini of AMS proteins are cytoplasmic and that processing of substrate peptides takes place at the cytosolic side of the membrane (7). A double glycine motif is present in the C-terminus of the leader ⁎ Corresponding author. Laboratory of Microbial Technology, Division of Microbial Science and Technology, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. Tel./fax: +81 92 642 3019. E-mail address: [email protected] (K. Sonomoto).
sequence of cognate prepeptide substrates of AMS proteins (6, 8). The first 150 amino acids from the N-terminus of some AMS proteins have been purified and shown to cleave off leader peptides in vitro; examples of such AMS proteins are LagD, transporter of the bacteriocin lactococcin G produced by Lactococcus lactis (6), CvaB (the transporter of colicin V produced by Escherichia coli (9)), and ComA (the transporter of the competence-stimulating peptide produced by Streptococcus pneumoniae (8)). Site-directed substitutions in conserved Cys and His residues of AMS proteins result in protein inactivation. These findings support the hypothesis that the N-terminal domain of AMS proteins is a papain-like cysteine protease (8, 9). Furgerson et al. (10) have reported the in vitro reconstitution of the activity of the N-terminal peptidase domain of LctT (lacticin 481 transporter) and have shown that the helical structure of the leader peptide is important for substrate recognition by this peptidase domain. The characteristics of the N-terminal domains of AMS proteins are being clarified. However, the functions of full-length AMS proteins remain unknown. Leader peptide cleavage is the final step of lantibiotic biosynthesis, and elucidation of the molecular functions of AMS proteins will clarify the molecular basis of lantibiotic biosynthesis. Nukacin ISK-1 is a type-A(II) lantibiotic produced by Staphyolococcus warneri ISK-1 (11–13). Ribosomally synthesized NukA consists of an N-terminal leader peptide and a C-terminal propeptide. The modification enzyme NukM introduces unusual amino acids in NukA to produce the modified NukA (14). NukT, a probable AMS protein, may contribute to leader peptide cleavage and mature nukacin ISK-1 transport (Fig. 1). We have previously reported that
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FIG. 1. Model of nukacin ISK-1 biosynthesis. Ribosomally synthesized prepeptide NukA consists of an N-terminal leader peptide and a C-terminal propeptide moiety. The modification enzyme NukM dehydrates serines and threonines and forms thioether rings by coupling dehydro residues to cysteines to produce modified NukA. The bifunctional ABC transporter NukT cleaves off leader peptide at a double glycine site to produce mature nukacin ISK-1. The darkly shaded residues represent modifiable and modified amino acids. A-S-A, lanthionine; Abu-S-A, 3-methyllanthionine; Dhb, dehydrobutyrine.
nukT inactivation blocks nukacin ISK-1 production, indicating that NukT is indispensable for nukacin ISK-1 biosynthesis (15). Here, we reconstituted in vitro the peptidase activity of full-length NukT overexpressed in inside-out membrane vesicles of Staphylococcus carnosus TM300. We found that NukT peptidase activity requires the presence of unusual amino acids in NukA and depends on ATP hydrolysis. This is the first in vitro study on lantibiotic processing by a full-length bifunctional ABC transporter. MATERIALS AND METHODS Molecular biology protocols Established molecular biology protocols were followed (16). PCR was performed with KOD Plus DNA polymerase (Toyobo, Osaka, Japan). The PCR products were purified with the Qiaquick PCR Purification Kit (Qiagen, West Sussex, UK). The restriction enzymes BamHI and NarI (Toyobo) were used according to the manufacturer's instructions. Staphylococcal plasmid DNA was prepared using Plasmid SV Mini (Geneall, Seoul, Korea), according to the manufacturer's instructions, except that the cells were incubated in 250 μl cellsuspension buffer containing 10 μg/ml lysostaphin (Wako, Osaka, Japan) for 30 min at 37 °C. nukT cloning A nukT fragment was amplified by PCR using pCnuk (template) (15) and the primers 5′-GTTACCGGATCCTCAGTTTGGAAGAAGGT-3′ and 5′-GTTAATCAGGCGCCAATCTCCTTAATTAT-3′. The amplified fragments were cloned into the BamHI and NarI sites of pTX15 (17); the resultant plasmid (pTXnukT) was introduced into S. carnosus TM300. S. carnosus TM300 transformation S. carnosus TM300 competent cells were prepared according to the method of Bringel et al. (18), with some modifications. In brief, S. carnosus TM300 was incubated overnight in 5 ml B2 broth (1.0% casein hydrolysate, 2.5% yeast extract, 0.1% K2HPO4, 0.5% glucose, 0.5% NaCl, pH adjusted to 7.5) at 37 °C. The cultured cells were transferred to 250 ml fresh B2 broth and incubated at 37 °C until an OD650 of 0.5–0.6, and the cells were harvested by centrifugation (6 500 × g, 15 min, 4 °C). The pellets obtained were washed twice with water and once with 10% glycerol, resuspended in 0.5 ml 10% glycerol, and immediately stored at − 80 °C. Plasmids (pTX15 or pTXnukT) were added to 70 μl of the above electrocompetent cell suspension. The suspension was transferred to a prechilled 0.2-cm gap cuvette and electroporated using a BioRad Gene Pulser (Bio-Rad, Hercules, CA) at 2.5 kV, 25 μF, and 200 Ω. The shocked cells were immediately resuspended in 390 μl B2 broth and incubated at 37 °C for 1 h. Next, 200 μl of cell suspension was spread on NYE agar plate
(1.0% casein hydrolysate, 0.5% yeast extract, 0.5% NaCl, 0.1% K2HPO4) containing 25 μg/ ml tetracycline and incubated overnight at 37 °C. Inside-out membrane vesicle preparation Inside-out membrane vesicles were prepared according to a previously described method (19), but with modifications. S. carnosus TM300 harboring pTX15 or pTXnukT was cultured at 37 °C for 18 h in 250 ml LB medium containing 0.5% xylose and 25 μg/ml tetracycline. The cultured cells were harvested by centrifugation (6500 × g, 15 min, 4 °C). The obtained pellets were washed with 50 mM K-HEPES buffer (pH 7.4); resuspended in K-HEPES buffer containing 5 μg/ml lysostaphin (Wako), 10 μg/ml DNase (Sigma, St. Louis, MO), 10 mM MgSO4, and Protease Inhibitor Cocktail (EDTA free, 100×; Nacalai Tesque, Kyoto, Japan); grown until an OD600 of 10; and incubated for 1 h at 37 °C. The cells were disrupted by 2 passages through a French Pressure Cells (Thermo Fisher Scientific, Waltham, MA) at 20,000 psi. Sodium-EDTA (pH 7.4) was added at a final concentration of 10 mM. Unbroken cells and cell debris were removed by centrifugation (6000 × g, 15 min, 4 °C). Inside-out membrane vesicles were harvested by ultra-centrifugation (210,000 × g, 1 h, 4 °C), washed with K-HEPES buffer, resuspended in 20 mM K-HEPES buffer (pH 7.4) containing 10% glycerol to a protein concentration of 5 mg/ml, and stored as 40-μl aliquots at − 80 °C. Protein concentrations were determined using a Bradford assay with bovine serum albumin (Sigma) as a standard. In vitro peptidase assay Modified His-tagged NukA (modified His–NukA) and His-tagged NukA (His–NukA) were prepared using previously described methods (14). NukT peptidase activity was assayed in a 30 μl reaction mixture containing 20 mM KHEPES buffer (pH 7.4), 5 mM DTT, 2 mM MgSO4, 5 mM ATP, and 3 μM modified His– NukA or His–NukA. The reaction was initiated by the addition of membrane vesicles (final protein concentration, 0.5 mg/ml) to the reaction mixture at 37 °C and stopped by cooling the mixture on ice. Tricine SDS-PAGE and overlay assay/western blotting The reaction mixture (10 μl) was subjected to 10% Tricine SDS-PAGE. After electrophoresis, the peptides were fixed in the gel by using a fixing solution (10% acetic acid/20% isopropanol) for 30 min. The gel was washed twice with distilled water for 30 min and incubated at 4 °C for 2 h. Nukacin ISK-1 activity was visualized using an overlay assay; the gel was transferred to an MRS agar plate (Oxoid, Hampshire, UK), overlaid with Lactobacilli agar AOAC (Difco Laboratories, Detroit, MI) seeded with Lactobacillus sakei subsp. sakei JCM 1157T as the indicator strain, and incubated overnight at 30 °C (20). The remaining substrates in the reaction mixture were visualized using western blotting with anti-histidine-tag antibody (Qiagen). L. sakei subsp. sakei JCM 1157T was cultured in MRS medium at 30 °C. MALDI-TOF mass spectrometry The membrane vesicles in the reaction mixture were collected by centrifugation (35,000 × g, 15 min, 4 °C) and resuspended in 30 μl of distilled water; 2 μl of suspension was applied to a detection plate and allowed to dry. Next, 2 μl of matrix (10 mg/ml α-cyano-4-hydroxycinnamic acid dissolved in 50%
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acetonitrile/0.1% trifluoroacetic acid) was added to the detection plate and allowed to dry. Mass spectra were recorded with the AXIMA-CFR and the mass spectrometer (Shimadzu Corporation, Kyoto, Japan and Kratos Analytical, Manchester, UK).
RESULTS NukT expression and localization in S. carnosus TM300 The gene encoding full-length NukT (694 amino acids) was cloned under the xylose-inducible promoter of pTX15. NukT was overexpressed in S. carnosus TM300 and cultivated in LB medium containing 0.5% xylose at 37 °C for 18 h. Cells were disrupted by two passages through French Pressure Cells. The membrane and cytoplasmic fractions were separated by ultra-centrifugation. Western blotting with anti-NukT antiserum showed that signals corresponding to NukT (80 kDa) were observed only in the case of the membrane fraction (data not shown). These results are consistent with those obtained using the nukacin ISK-1-producing strain, S. warneri ISK-1 (21); this indicates that the localization of NukT in S. carnosus TM300 was the same as that observed in case of S. warneri ISK-1. NukT-mediated leader peptide cleavage in vitro To assess the NukT peptidase activity in vitro, we prepared inside-out membrane vesicles expressing NukT (NukT membrane). Since the N-terminal peptidase domain of NukT is probably intracytoplasmic, inside-out membrane vesicles were used. In addition, N-terminal histidine-tag fusion of prepeptide NukA containing unusual amino acids (modified His–NukA) was prepared using a system for the NukM-mediated introduction of unusual amino acids into NukA (14). NukT membrane vesicles were incubated with modified His–NukA in the presence of DTT, Mg2+, and ATP at 37 °C for 2 h. Reaction products were detected using an overlay assay; this assay enabled the monitoring of bioactive peptides by inhibiting growth of the indicator strain. On reaction with the NukT membrane, the reactants produced inhibition zones and exhibited the same electrophoretic mobility as purified nukacin ISK-1 (Fig. 2A, lanes 3 and 4). The reaction mixture was then subjected to MALDI-TOF mass spectrometry to confirm the molecular weight of the product. The membrane fraction was harvested to collect peptides and roughly remove salts from the reaction mixture. The molecular weight of the peptide produced by NukT membrane was 2960 Da, which corre-
FIG. 3. Effects of protease inhibitors on NukT peptidase activity. Modified His–NukA (3 μM) was incubated with 0.5 mg/ml NukT membrane in the presence of protease inhibitors. Nukacin ISK-1 activity was visualized as in Fig. 2A. Lane 1, without protease inhibitor; lane 2, 1 mM PMSF (serine/cysteine protease inhibitor); lane 3, 5.6 mM E-64 (serine/cysteine protease inhibitor); lane 4, 14 μM pepstatin (aspartic acid protease inhibitor); lane 5, 3.5 mM phosphoramidon (metalloprotease inhibitor); and lane 6, 5 mM EDTA (metalloprotease inhibitor). The arrow indicates the position of nukacin ISK-1.
sponds to that of nukacin ISK-1; thus, the bioactive peptide observed on SDS-PAGE was nukacin ISK-1 (Fig. 2B). Control membrane prepared from S. carnosus harboring pTX15 did not produce an inhibition zone (Fig. 2A, lane 2); this excluded the possibility of leader peptide cleavage by a nonspecific protease. We also analyzed the unreacted substrates remaining in the reaction mixture by western blotting with an anti-histidine-tag antibody. The signal corresponding to modified His–NukA completely disappeared after incubation with NukT membrane but not after incubation with the control membrane (Fig. 2C, lanes 1 and 2). This indicates that NukT cleaves off the leader peptide of modified His–NukA at the double glycine cleavage site to produce mature nukacin ISK-1. Unexpectedly, no signals corresponding to the leader peptide were detected in the reaction mixture by western blotting and MALDI-TOF mass spectrometry. NukT substrate specificity To identify the substrates on which NukT exerts its peptidase activity, we used the unmodified prepeptide His–NukA, which does not contain any unusual amino acids. After the reaction, the untreated substrates remaining in the reaction mixture
FIG. 2. Leader peptide cleavage by NukT. Modified His–NukA or unmodified His–NukA (3 μM) was incubated with/without 0.5 mg/ml inside-out membrane vesicles of Staphylococcus carnosus TM300 harboring pTXnukT (NukT membrane) or pTX15 (control membrane) in 30 μl of reaction mixture containing 20 mM K-HEPES buffer (pH 7.4), 5 mM DTT, 2 mM MgCl2, and 5 mM ATP at 37 °C for 2 h. The reaction mixture (10 μl) was subjected to 10% Tricine SDS-PAGE. (A) Nukacin ISK-1 activity was visualized using an overlay assay, as described in Materials and methods, with Lactobacillus sakei subsp. sakei JCM 1157T as the indicator strain. Lane 1, modified His–NukA; lane 2, modified His–NukA incubated with control membrane; lane 3, modified His–NukA incubated with NukT membrane; and lane 4, 3 μM purified nukacin ISK-1. The arrow indicates the position of nukacin ISK-1. (B) MALDI-TOF mass spectrum after incubation of modified His–NukA with NukT membrane. A peak corresponding to nukacin ISK-1 (2960 Da) is detected. (C) The remaining substrates in the reaction mixture were visualized using western blotting with anti-histidine-tag antibody. Lane 1, modified His–NukA incubated with control membrane; lane 2, modified His– NukA incubated with NukT membrane; lane 3, His–NukA incubated with control membrane; and lane 4, His–NukA incubated with NukT membrane. The black and white arrowheads indicate the position of modified His–NukA and unmodified His–NukA, respectively.
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FIG. 4. Effects of ATP on NukT peptidase activity. Lane 1, 3 μM nukacin ISK-1. Modified His–NukA (3 μM) and NukT membrane (0.5 mg/ml) were incubated with 5 mM ATP (lane 2), without ATP (lane 3), with 5 mM ATP-γ-S (non-hydrolyzable ATP analogue; lane 4), and with 5 mM ATP and 5 mM sodium orthovanadate (ATPase inhibitor; lane 5). Nukacin ISK-1 activity was visualized as in Fig. 2A. The arrow indicates the position of nukacin ISK-1.
were analyzed by western blotting. In contrast to the result obtained using modified His–NukA as a substrate, the signal corresponding to His–NukA remained visible after 2-h reactions with NukT membrane (Fig. 2C, lane 4) and control membrane (Fig. 2C, lane 3); this indicated that NukT does not process His–NukA and that the unusual amino acids in NukA are required for substrate recognition by NukT. Effects of protease inhibitors The similarity between the sequences of NukT and other AMS proteins suggests that the Nterminal domain of NukT is a cysteine protease (6). To characterize NukT-mediated leader peptide cleavage, we determined the effects of the inhibitors of serine/cysteine protease (1 mM PMSF and 5.6 mM E64), aspartic acid protease (14 μM pepstatin), and metalloprotease (3.5 mM phosphoramidon and 5 mM EDTA) on NukT peptidase activity using an in vitro peptidase assay followed by Tricine SDS-PAGE and an overlay assay (described in Materials and methods). PMSF inhibited nukacin ISK-1 production (Fig. 3, lane 2), whereas E-64, pepstatin, and phosphoramidon did not affect NukT peptidase activity (Fig. 3, lanes 3–5). This result supports the sequence prediction that NukT is a cysteine protease. It should be noted that phosphoramidon did not affect NukT peptidase activity, but EDTA did (Fig. 3, lane 6), suggesting that, although NukT is not a metalloprotease, some metal ions may support NukT peptidase activity. Effect of ATP hydrolysis It is generally accepted that the ATPase activity of ABC transporters supplies the energy required for ABC protein-dependent transport. The C-terminal domain of NukT is predicted to be an ATP-binding domain; we examined the effect of ATP hydrolysis on NukT peptidase activity using an in vitro peptidase assay followed by Tricine SDS-PAGE and an overlay assay. Unlike the reaction mixture containing 5 mM ATP, the ATP-free mixture did not exhibit peptidase activity (Fig. 4, lanes 2 and 3). NukT-mediated processing was completely abolished by substituting ATP with 5 mM ATP-γ-s (a non-hydrolyzable ATP analogue) (Fig. 4, lane 4). Additionally, 5 mM vanadate (an ATPase inhibitor) inhibited the NukT activity in the presence of ATP (Fig. 4, lane 5). The unreacted substrates in the reaction mixtures that did not exhibit nukacin ISK-1 activity were detected by western blotting (data not shown). These results suggest that ATP hydrolysis provides sufficient energy for NukT peptidase activity. Furthermore, this activity was not observed in the absence of Mg2+ (data not shown), indicating that Mg2+ is an essential co-factor of NukT. DISCUSSION NukT (a probable AMS protein) is a bifunctional protein that is predicted to mediate leader peptide cleavage and export of type-A(II) lantibiotic nukacin ISK-1. Generally, it is difficult to express membrane proteins from gram-positive bacteria in the gram-negative E. coli; for example, attempts to express full-length ComA (an AMS protein from S. pneumoniae) in E. coli have failed (8). Only the N-terminal peptidase
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domains (ca. 150 amino acids) of some AMS proteins from grampositive bacteria have been expressed in E. coli, and the peptidase activity of these proteins has been characterized (6, 8, 10). Recently, in vitro reconstitution of the peptidase activity of the N-terminal domain of LctT was reported; LctT transports lacticin 481, which is classified under the same type of lantibiotics as nukacin ISK-1 (10). In this study, we successfully expressed full-length NukT in S. carnosus TM300. To characterize the peptidase activity of full-length NukT, we first developed an in vitro assay using inside-out membrane vesicles of S. carnosus expressing NukT. We found that NukT cleaves off the leader peptide of modified His– NukA containing unusual amino acids, but it did not affect leader peptide cleavage of unmodified His–NukA (Fig. 2). This result suggests that the unusual amino acids in NukA are required for substrate recognition by NukT and may also inhibit cleavage of the C-terminal propeptide moiety after recognition by NukT. It has been reported that the first 150 amino acids of LctT can process LctM-treated LctA prepeptide containing three lanthionine rings with similar efficacy to unmodified LctA; this indicates that the rings are not responsible for substrate recognition by the N-terminal domain of LctT (10). On the other hand, the peptidase activity of NisP (a serine protease involved in processing of the leader peptide of type-A(I) lantibiotic nisin) depends on the thioether rings in nisin prepeptide (22). NukT and LctT are AMS proteins involved in the biosynthesis of type-A(II) lantibiotics and exhibit 49% similarity (full-length proteins); their N-terminal peptidase domains show 45% similarity. The substrate specificity of NukT was different from that of the N-terminal domain of LctT but is otherwise poorly understood. At present, we have no data regarding substrate recognition rules for the N-terminal domain of NukT. On the basis of our data, we propose that the substrate recognition mechanism of full-length NukT differs from that of its N-terminal peptidase domain. We found that signals corresponding to the leader peptide were not detected after the in vitro reaction. In contrast, the study of ComA and LctT detected the leader peptides of their respective substrates after the reaction (8, 10). The leader peptide is small and linear and may therefore be easily digested into small fragments by nonspecific proteases present in inside-out membrane vesicles after being processed by NukT or by a specific protease activity of NukT. Sequence comparisons revealed that the Cys12 and His90 of NukT were completely conserved in other cysteine proteases; this suggested that NukT, like other AMS proteins, is a papain-like cysteine protease (6). In this study, the peptidase activity of NukT was completely inhibited by the serine/cysteine protease inhibitor PMSF (Fig. 3), thus strongly supporting the sequence prediction. Surprisingly, E-64, which is also a serine/cysteine protease inhibitor, did not inhibit NukT peptidase activity. Similarly, the N-terminal domain of ComA was not sensitive to E-64 (8). Since AMS proteins exhibit greater substrate specificity than protein-degrading proteases, NukT or the Nterminal domain of ComA might tolerate typical peptide-mimetic serine/cysteine protease inhibitors. One of the interesting findings in this study is that NukT peptidase activity required ATP hydrolysis (Fig. 4). NukT did not cleave off leader peptide without ATP or in the presence of ATP-γ-S (instead of ATP) or vanadate (an ATPase inhibitor). This suggested that ATP hydrolysis provides sufficient energy for NukT peptidase activity. In contrast, the N-terminal peptidase domains of AMS proteins such as LagD, ComA, CvaB, and LctT do not require ATP for their peptidase activity (6, 8–10). The peptidase activity of full-length CvaB (a specific colicin V exporter and an AMS protein produced by gram-negative bacteria) has been characterized. Zhong et al. (23) have suggested that the processing of colicin V precursor by full-length CvaB requires an intact CvaA–CvaB– TolC complex and energy in the form of nucleotide hydrolysis (23). Furthermore, nucleotide binding was required for CvaB dimerization (24). ABC transporters require ATP to couple the hydrolysis of ATP to
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the translocation of solutes across a biological membrane. ATP hydrolysis is coupled to conformational changes in the transporter that mediate the movement of substrate across the membrane (25). Clp, Lon, and FtsH proteases are ATP-dependent proteases that use the free energy of ATP hydrolysis to unfold protein substrates and present them successively to protease active sites. Crystallographic and biochemical studies of these proteins have proposed that the ATPase module unfolds, translocates, and activates proteolytic sites through conformational changes induced by repetitive ATPase cycles (26–28). As one possibility, NukT may utilize the energy of ATP hydrolysis to activate proteolytic sites through conformational changes, and this may determine the substrate specificity of its peptidase domain. Besides ATP, NukT also required Mg2+ for its peptidase activity (data not shown), which explains why EDTA inhibited NukT peptidase activity (Fig. 3, lane 6). Mg2+ is important for ATPase to orient all residues active in catalysis in a productive formation (29); therefore, Mg2+ chelation by EDTA would result in NukT inactivation. While the N-terminal domain of CvaB requires Ca2+ as a co-factor, NukT did not (data not shown). NukT is comparable to the N-terminal domain of ComA or LctT, both of which do not require Ca2+ (8, 10). In summary, full-length NukT cleaves off the leader peptide of modified His–NukA in vitro. NukT may be a cysteine protease; its peptidase activity depends on ATP hydrolysis and the presence of unusual amino acids in NukA. This is the first study to report in detail the peptidase activity of LanT of a type-A(II) lantibiotic. It should be emphasized that our findings provide a novel insight into the molecular basis of lantibiotic biosynthesis and will facilitate the biological production of the engineered lantibiotics. Further studies on full-length NukT are required to provide an understanding of the characteristics of AMS proteins. ACKNOWLEDGMENTS We are grateful to F. Götz (University of Tübingen) for providing S. carnosus TM300 and pTX15. This study was partially supported by grants from the Japan Society for the Promotion of Science (JSPS), Japan Science Society, Novartis Foundation (Japan) for the Promotion of Science, Novozymes Japan Research Fund, and Nagase Science and Technology Foundation. References 1. de Vos, W. M., Kuipers, O. P., van der Meer, J. R., and Siezen, R. J.: Maturation pathway of nisin and other lantibiotics: post-translationally modified antimicrobial peptides exported by gram-positive bacteria, Mol. Microbiol., 17, 427–437 (1995). 2. Sahl, H.-G., Jack, R. W., and Bierbaum, G.: Biosynthesis and biological activities of lantibiotics with unique post-translational modifications, Eur. J. Biochem., 230, 827–853 (1995). 3. McAuliffe, O., Ross, R. P., and Hill, C.: Lantibiotics: structure, biosynthesis and mode of action, FEMS Microbiol. Rev., 25, 285–308 (2001). 4. Chatterjee, C., Paul, M., Xie, L., and van der Donk, W. A.: Biosynthesis and mode of action of lantibiotics, Chem. Rev., 105, 633–684 (2005). 5. Dufour, A., Hindre, T., Haras, D., and Le Pennec, J. P.: The biology of lantibiotics from the lacticin 481 group is coming of age, FEMS Microbiol. Rev., 31, 134–167 (2007). 6. Havarstein, L. S., Diep, D. B., and Nes, I. F.: A family of bacteriocin ABC transporters carry out proteolytic processing of their substrates concomitant with export, Mol. Microbiol., 16, 229–240 (1995). 7. Franke, C. M., Tiemersma, J., Venema, G., and Kok, J.: Membrane topology of the lactococcal bacteriocin ATP-binding cassette transporter protein LcnC. Involvement of LcnC in lactococcin maturation, J. Biol. Chem., 274, 8484–8490 (1999).
J. BIOSCI. BIOENG., 8. Ishii, S., Yano, T., and Hayashi, H.: Expression and characterization of the peptidase domain of Streptococcus pneumoniae ComA, a bifunctional ATP-binding cassette transporter involved in quorum sensing pathway, J. Biol. Chem., 281, 4726–4731 (2006). 9. Wu, K. H. and Tai, P. C.: Cys32 and His105 are the critical residues for the calciumdependent cysteine proteolytic activity of CvaB, an ATP-binding cassette transporter, J. Biol. Chem., 279, 901–909 (2004). 10. Furgerson Ihnken, L. A., Chatterjee, C., and van der Donk, W. A.: In vitro reconstitution and substrate specificity of a lantibiotic protease, Biochemistry, 47, 7352–7363 (2008). 11. Kimura, H., Nagano, R., Matsusaki, H., Sonomoto, K., and Ishizaki, A.: A bacteriocin of strain Pediococcus sp. ISK-1 isolated from Nukadoko, bed of fermented rice bran, Biosci. Biotechnol. Biochem., 61, 1049–1051 (1997). 12. Kimura, H., Sashihara, T., Matsusaki, H., Sonomoto, K., and Ishizaki, A.: Novel bacteriocin of Pediococcus sp. ISK-1 isolated from well-aged bed of fermented rice bran, Ann. N.Y. Acad. Sci., 864, 345–348 (1998). 13. Sashihara, T., Kimura, H., Higuchi, T., Adachi, A., Matsusaki, H., Sonomoto, K., and Ishizaki, A.: A novel lantibiotic, nukacin ISK-1, of Staphylococcus warneri ISK-1: cloning of the structural gene and identification of the structure, Biosci. Biotechnol. Biochem., 64, 2420–2428 (2000). 14. Nagao, J., Harada, Y., Shioya, K., Aso, Y., Zendo, T., Nakayama, J., and Sonomoto, K.: Lanthionine introduction into nukacin ISK-1 prepeptide by co-expression with modification enzyme NukM in Escherichia coli, Biochem. Biophys. Res. Commun., 336, 507–513 (2005). 15. Aso, Y., Nagao, J., Koga, H., Okuda, K., Kanemasa, Y., Sashihara, T., Nakayama, J., and Sonomoto, K.: Heterologous expression and functional analysis of the gene cluster for the biosynthesis of and immunity to the lantibiotic, nukacin ISK-1, J. Biosci. Bioeng., 98, 429–436 (2004). 16. Sambrook, J., Fritsch, E., and Maniatis, T.: Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, New York, 1989. 17. Peschel, A., Ottenwalder, B., and Gotz, F.: Inducible production and cellular location of the epidermin biosynthetic enzyme EpiB using an improved staphylococcal expression system, FEMS Microbiol. Lett., 137, 279–284 (1996). 18. Bringel, F. and Hubert, J.-C.: Optimized transformation by electroporation of Lactobacillus plantarum strains with plasmid vectors, Appl. Microbiol. Biotechnol., 33, 664–670 (1990). 19. Reuter, G., Janvilisri, T., Venter, H., Shahi, S., Balakrishnan, L., and van Veen, H. W.: The ATP binding cassette multidrug transporter LmrA and lipid transporter MsbA have overlapping substrate specificities, J. Biol. Chem., 278, 35193–35198 (2003). 20. Bhunia, A. K., Johnson, M. C., and Ray, B.: Direct detection of an antimicrobial peptide of Pediococcus acicilactici in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, J. Ind. Microbiol., 2, 319–322 (1987). 21. Nagao, J., Aso, Y., Sashihara, T., Shioya, K., Adachi, A., Nakayama, J., and Sonomoto, K.: Localization and interaction of the biosynthetic proteins for the lantibiotic, Nukacin ISK-1, Biosci. Biotechnol. Biochem., 69, 1341–1347 (2005). 22. Kuipers, A., de Boef, E., Rink, R., Fekken, S., Kluskens, L. D., Driessen, A. J., Leenhouts, K., Kuipers, O. P., and Moll, G. N.: NisT, the transporter of the lantibiotic nisin, can transport fully modified, dehydrated, and unmodified prenisin and fusions of the leader peptide with non-lantibiotic peptides, J. Biol. Chem., 279, 22176–22182 (2004). 23. Zhong, X., Kolter, R., and Tai, P. C.: Processing of colicin V-1, a secretable marker protein of a bacterial ATP binding cassette export system, requires membrane integrity, energy, and cytosolic factors, J. Biol. Chem., 271, 28057–28063 (1996). 24. Guo, X., Harrison, R. W., and Tai, P. C.: Nucleotide-dependent dimerization of the C-terminal domain of the ABC transporter CvaB in colicin V secretion, J. Bacteriol., 188, 2383–2391 (2006). 25. Davidson, A. L. and Chen, J.: ATP-binding cassette transporters in bacteria, Annu. Rev. Biochem., 73, 241–268 (2004). 26. Wang, J., Song, J. J., Franklin, M. C., Kamtekar, S., Im, Y. J., Rho, S. H., Seong, I. S., Lee, C. S., Chung, C. H., and Eom, S. H.: Crystal structures of the HslVU peptidaseATPase complex reveal an ATP-dependent proteolysis mechanism, Structure, 9, 177–184 (2001). 27. Hinnerwisch, J., Fenton, W. A., Furtak, K. J., Farr, G. W., and Horwich, A. L.: Loops in the central channel of ClpA chaperone mediate protein binding, unfolding, and translocation, Cell, 121, 1029–1041 (2005). 28. Licht, S. and Lee, I.: Resolving individual steps in the operation of ATP-dependent proteolytic molecular machines: from conformational changes to substrate translocation and processivity, Biochemistry, 47, 3595–3605 (2008). 29. Davidson, A. L., Dassa, E., Orelle, C., and Chen, J.: Structure, function, and evolution of bacterial ATP-binding cassette systems, Microbiol. Mol. Biol. Rev., 72, 317–364 (2008).
ATP-dependent leader peptide cleavage by NukT, a bifunctional ABC transporter, during lantibiotic biosynthesis Mami Nishie,1 Kouki Shioya,1 Jun-ichi Nagao,2 Hiroyuki Jikuya,3 and Kenji Sonomoto1,3,⁎ Laboratory of Microbial Technology, Division of Microbial Science and Technology, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan 1 Department of Functional Bioscience, Section of Infection Biology, Fukuoka Dental College, 2-15-1 Tamura, Sawara-ku, Fukuoka 814-0193, Japan 2 and Bio-Architecture Center, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan 3 Received 30 April 2009; accepted 1 June 2009
NukT, a possible ABC transporter maturation and secretion (AMS) protein, may contribute to the cleavage of the leader peptide of NukA, which is the prepeptide of the lantibiotic nukacin ISK-1, and to nukacin ISK-1 transport. In this study, we reconstituted in vitro peptidase activity of the full-length NukT overexpressed in inside-out membrane vesicles of Staphylococcus carnosus TM300. We found that the presence of unusual amino acids in NukA is required for leader peptide cleavage. Furthermore, NukT peptidase activity was inhibited by phenylmethylsulfonyl fluoride, a serine/cysteine protease inhibitor; this finding strongly suggests that NukT, like other AMS proteins, is a cysteine protease. Interestingly, NukT peptidase activity depended on ATP hydrolysis. These results suggest that the N-terminal peptidase domain of NukT may cooperatively function with the C-terminal ATP-binding domain. This is the first in vitro study on lantibiotics that reports the processing mechanism of a full-length bifunctional ABC transporter. © 2009, The Society for Biotechnology, Japan. All rights reserved. [Key words: Lantibiotic; Post-translational modification; Peptidase; ABC transporter]
Lantibiotics, antimicrobial peptides produced by gram-positive bacteria, contain unusual (e.g., lanthionine) and dehydrated amino acids. Ribosomally synthesized lantibiotic prepeptides consist of an Nterminal leader peptide and a C-terminal propeptide that undergoes several post-translational modifications (1, 2). Type-A(I) lantibiotics (e.g., nisin) undergo the following modifications: LanB dehydrates serines and threonines, LanC forms thioether rings (involving coupling of dehydro residues to cysteins), LanT exports modified prepeptides, and LanP cleaves off N-terminal leader peptides from exported peptides (LanP is an extracellular serine protease) (1, 3, 4). Type-A(II) lantibiotics (e.g., lacticin 481) undergo LanM-mediated dehydration and cyclization, which results in the formation of unusual amino acids, LanTmediated cleavage of the N-terminal leader peptide (LanT is a bifunctional ABC transporter) and secretion of mature lantibiotics (5). LanT, specific to type-A(II) lantibiotics, is an ABC transporter maturation and secretion (AMS) protein; AMS proteins comprise an N-terminal putative peptidase domain, a transmembrane domain, and a C-terminal ATP-binding domain (6). It is predicted that the N- and Ctermini of AMS proteins are cytoplasmic and that processing of substrate peptides takes place at the cytosolic side of the membrane (7). A double glycine motif is present in the C-terminus of the leader ⁎ Corresponding author. Laboratory of Microbial Technology, Division of Microbial Science and Technology, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. Tel./fax: +81 92 642 3019. E-mail address: [email protected] (K. Sonomoto).
sequence of cognate prepeptide substrates of AMS proteins (6, 8). The first 150 amino acids from the N-terminus of some AMS proteins have been purified and shown to cleave off leader peptides in vitro; examples of such AMS proteins are LagD, transporter of the bacteriocin lactococcin G produced by Lactococcus lactis (6), CvaB (the transporter of colicin V produced by Escherichia coli (9)), and ComA (the transporter of the competence-stimulating peptide produced by Streptococcus pneumoniae (8)). Site-directed substitutions in conserved Cys and His residues of AMS proteins result in protein inactivation. These findings support the hypothesis that the N-terminal domain of AMS proteins is a papain-like cysteine protease (8, 9). Furgerson et al. (10) have reported the in vitro reconstitution of the activity of the N-terminal peptidase domain of LctT (lacticin 481 transporter) and have shown that the helical structure of the leader peptide is important for substrate recognition by this peptidase domain. The characteristics of the N-terminal domains of AMS proteins are being clarified. However, the functions of full-length AMS proteins remain unknown. Leader peptide cleavage is the final step of lantibiotic biosynthesis, and elucidation of the molecular functions of AMS proteins will clarify the molecular basis of lantibiotic biosynthesis. Nukacin ISK-1 is a type-A(II) lantibiotic produced by Staphyolococcus warneri ISK-1 (11–13). Ribosomally synthesized NukA consists of an N-terminal leader peptide and a C-terminal propeptide. The modification enzyme NukM introduces unusual amino acids in NukA to produce the modified NukA (14). NukT, a probable AMS protein, may contribute to leader peptide cleavage and mature nukacin ISK-1 transport (Fig. 1). We have previously reported that
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FIG. 1. Model of nukacin ISK-1 biosynthesis. Ribosomally synthesized prepeptide NukA consists of an N-terminal leader peptide and a C-terminal propeptide moiety. The modification enzyme NukM dehydrates serines and threonines and forms thioether rings by coupling dehydro residues to cysteines to produce modified NukA. The bifunctional ABC transporter NukT cleaves off leader peptide at a double glycine site to produce mature nukacin ISK-1. The darkly shaded residues represent modifiable and modified amino acids. A-S-A, lanthionine; Abu-S-A, 3-methyllanthionine; Dhb, dehydrobutyrine.
nukT inactivation blocks nukacin ISK-1 production, indicating that NukT is indispensable for nukacin ISK-1 biosynthesis (15). Here, we reconstituted in vitro the peptidase activity of full-length NukT overexpressed in inside-out membrane vesicles of Staphylococcus carnosus TM300. We found that NukT peptidase activity requires the presence of unusual amino acids in NukA and depends on ATP hydrolysis. This is the first in vitro study on lantibiotic processing by a full-length bifunctional ABC transporter. MATERIALS AND METHODS Molecular biology protocols Established molecular biology protocols were followed (16). PCR was performed with KOD Plus DNA polymerase (Toyobo, Osaka, Japan). The PCR products were purified with the Qiaquick PCR Purification Kit (Qiagen, West Sussex, UK). The restriction enzymes BamHI and NarI (Toyobo) were used according to the manufacturer's instructions. Staphylococcal plasmid DNA was prepared using Plasmid SV Mini (Geneall, Seoul, Korea), according to the manufacturer's instructions, except that the cells were incubated in 250 μl cellsuspension buffer containing 10 μg/ml lysostaphin (Wako, Osaka, Japan) for 30 min at 37 °C. nukT cloning A nukT fragment was amplified by PCR using pCnuk (template) (15) and the primers 5′-GTTACCGGATCCTCAGTTTGGAAGAAGGT-3′ and 5′-GTTAATCAGGCGCCAATCTCCTTAATTAT-3′. The amplified fragments were cloned into the BamHI and NarI sites of pTX15 (17); the resultant plasmid (pTXnukT) was introduced into S. carnosus TM300. S. carnosus TM300 transformation S. carnosus TM300 competent cells were prepared according to the method of Bringel et al. (18), with some modifications. In brief, S. carnosus TM300 was incubated overnight in 5 ml B2 broth (1.0% casein hydrolysate, 2.5% yeast extract, 0.1% K2HPO4, 0.5% glucose, 0.5% NaCl, pH adjusted to 7.5) at 37 °C. The cultured cells were transferred to 250 ml fresh B2 broth and incubated at 37 °C until an OD650 of 0.5–0.6, and the cells were harvested by centrifugation (6 500 × g, 15 min, 4 °C). The pellets obtained were washed twice with water and once with 10% glycerol, resuspended in 0.5 ml 10% glycerol, and immediately stored at − 80 °C. Plasmids (pTX15 or pTXnukT) were added to 70 μl of the above electrocompetent cell suspension. The suspension was transferred to a prechilled 0.2-cm gap cuvette and electroporated using a BioRad Gene Pulser (Bio-Rad, Hercules, CA) at 2.5 kV, 25 μF, and 200 Ω. The shocked cells were immediately resuspended in 390 μl B2 broth and incubated at 37 °C for 1 h. Next, 200 μl of cell suspension was spread on NYE agar plate
(1.0% casein hydrolysate, 0.5% yeast extract, 0.5% NaCl, 0.1% K2HPO4) containing 25 μg/ ml tetracycline and incubated overnight at 37 °C. Inside-out membrane vesicle preparation Inside-out membrane vesicles were prepared according to a previously described method (19), but with modifications. S. carnosus TM300 harboring pTX15 or pTXnukT was cultured at 37 °C for 18 h in 250 ml LB medium containing 0.5% xylose and 25 μg/ml tetracycline. The cultured cells were harvested by centrifugation (6500 × g, 15 min, 4 °C). The obtained pellets were washed with 50 mM K-HEPES buffer (pH 7.4); resuspended in K-HEPES buffer containing 5 μg/ml lysostaphin (Wako), 10 μg/ml DNase (Sigma, St. Louis, MO), 10 mM MgSO4, and Protease Inhibitor Cocktail (EDTA free, 100×; Nacalai Tesque, Kyoto, Japan); grown until an OD600 of 10; and incubated for 1 h at 37 °C. The cells were disrupted by 2 passages through a French Pressure Cells (Thermo Fisher Scientific, Waltham, MA) at 20,000 psi. Sodium-EDTA (pH 7.4) was added at a final concentration of 10 mM. Unbroken cells and cell debris were removed by centrifugation (6000 × g, 15 min, 4 °C). Inside-out membrane vesicles were harvested by ultra-centrifugation (210,000 × g, 1 h, 4 °C), washed with K-HEPES buffer, resuspended in 20 mM K-HEPES buffer (pH 7.4) containing 10% glycerol to a protein concentration of 5 mg/ml, and stored as 40-μl aliquots at − 80 °C. Protein concentrations were determined using a Bradford assay with bovine serum albumin (Sigma) as a standard. In vitro peptidase assay Modified His-tagged NukA (modified His–NukA) and His-tagged NukA (His–NukA) were prepared using previously described methods (14). NukT peptidase activity was assayed in a 30 μl reaction mixture containing 20 mM KHEPES buffer (pH 7.4), 5 mM DTT, 2 mM MgSO4, 5 mM ATP, and 3 μM modified His– NukA or His–NukA. The reaction was initiated by the addition of membrane vesicles (final protein concentration, 0.5 mg/ml) to the reaction mixture at 37 °C and stopped by cooling the mixture on ice. Tricine SDS-PAGE and overlay assay/western blotting The reaction mixture (10 μl) was subjected to 10% Tricine SDS-PAGE. After electrophoresis, the peptides were fixed in the gel by using a fixing solution (10% acetic acid/20% isopropanol) for 30 min. The gel was washed twice with distilled water for 30 min and incubated at 4 °C for 2 h. Nukacin ISK-1 activity was visualized using an overlay assay; the gel was transferred to an MRS agar plate (Oxoid, Hampshire, UK), overlaid with Lactobacilli agar AOAC (Difco Laboratories, Detroit, MI) seeded with Lactobacillus sakei subsp. sakei JCM 1157T as the indicator strain, and incubated overnight at 30 °C (20). The remaining substrates in the reaction mixture were visualized using western blotting with anti-histidine-tag antibody (Qiagen). L. sakei subsp. sakei JCM 1157T was cultured in MRS medium at 30 °C. MALDI-TOF mass spectrometry The membrane vesicles in the reaction mixture were collected by centrifugation (35,000 × g, 15 min, 4 °C) and resuspended in 30 μl of distilled water; 2 μl of suspension was applied to a detection plate and allowed to dry. Next, 2 μl of matrix (10 mg/ml α-cyano-4-hydroxycinnamic acid dissolved in 50%
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acetonitrile/0.1% trifluoroacetic acid) was added to the detection plate and allowed to dry. Mass spectra were recorded with the AXIMA-CFR and the mass spectrometer (Shimadzu Corporation, Kyoto, Japan and Kratos Analytical, Manchester, UK).
RESULTS NukT expression and localization in S. carnosus TM300 The gene encoding full-length NukT (694 amino acids) was cloned under the xylose-inducible promoter of pTX15. NukT was overexpressed in S. carnosus TM300 and cultivated in LB medium containing 0.5% xylose at 37 °C for 18 h. Cells were disrupted by two passages through French Pressure Cells. The membrane and cytoplasmic fractions were separated by ultra-centrifugation. Western blotting with anti-NukT antiserum showed that signals corresponding to NukT (80 kDa) were observed only in the case of the membrane fraction (data not shown). These results are consistent with those obtained using the nukacin ISK-1-producing strain, S. warneri ISK-1 (21); this indicates that the localization of NukT in S. carnosus TM300 was the same as that observed in case of S. warneri ISK-1. NukT-mediated leader peptide cleavage in vitro To assess the NukT peptidase activity in vitro, we prepared inside-out membrane vesicles expressing NukT (NukT membrane). Since the N-terminal peptidase domain of NukT is probably intracytoplasmic, inside-out membrane vesicles were used. In addition, N-terminal histidine-tag fusion of prepeptide NukA containing unusual amino acids (modified His–NukA) was prepared using a system for the NukM-mediated introduction of unusual amino acids into NukA (14). NukT membrane vesicles were incubated with modified His–NukA in the presence of DTT, Mg2+, and ATP at 37 °C for 2 h. Reaction products were detected using an overlay assay; this assay enabled the monitoring of bioactive peptides by inhibiting growth of the indicator strain. On reaction with the NukT membrane, the reactants produced inhibition zones and exhibited the same electrophoretic mobility as purified nukacin ISK-1 (Fig. 2A, lanes 3 and 4). The reaction mixture was then subjected to MALDI-TOF mass spectrometry to confirm the molecular weight of the product. The membrane fraction was harvested to collect peptides and roughly remove salts from the reaction mixture. The molecular weight of the peptide produced by NukT membrane was 2960 Da, which corre-
FIG. 3. Effects of protease inhibitors on NukT peptidase activity. Modified His–NukA (3 μM) was incubated with 0.5 mg/ml NukT membrane in the presence of protease inhibitors. Nukacin ISK-1 activity was visualized as in Fig. 2A. Lane 1, without protease inhibitor; lane 2, 1 mM PMSF (serine/cysteine protease inhibitor); lane 3, 5.6 mM E-64 (serine/cysteine protease inhibitor); lane 4, 14 μM pepstatin (aspartic acid protease inhibitor); lane 5, 3.5 mM phosphoramidon (metalloprotease inhibitor); and lane 6, 5 mM EDTA (metalloprotease inhibitor). The arrow indicates the position of nukacin ISK-1.
sponds to that of nukacin ISK-1; thus, the bioactive peptide observed on SDS-PAGE was nukacin ISK-1 (Fig. 2B). Control membrane prepared from S. carnosus harboring pTX15 did not produce an inhibition zone (Fig. 2A, lane 2); this excluded the possibility of leader peptide cleavage by a nonspecific protease. We also analyzed the unreacted substrates remaining in the reaction mixture by western blotting with an anti-histidine-tag antibody. The signal corresponding to modified His–NukA completely disappeared after incubation with NukT membrane but not after incubation with the control membrane (Fig. 2C, lanes 1 and 2). This indicates that NukT cleaves off the leader peptide of modified His–NukA at the double glycine cleavage site to produce mature nukacin ISK-1. Unexpectedly, no signals corresponding to the leader peptide were detected in the reaction mixture by western blotting and MALDI-TOF mass spectrometry. NukT substrate specificity To identify the substrates on which NukT exerts its peptidase activity, we used the unmodified prepeptide His–NukA, which does not contain any unusual amino acids. After the reaction, the untreated substrates remaining in the reaction mixture
FIG. 2. Leader peptide cleavage by NukT. Modified His–NukA or unmodified His–NukA (3 μM) was incubated with/without 0.5 mg/ml inside-out membrane vesicles of Staphylococcus carnosus TM300 harboring pTXnukT (NukT membrane) or pTX15 (control membrane) in 30 μl of reaction mixture containing 20 mM K-HEPES buffer (pH 7.4), 5 mM DTT, 2 mM MgCl2, and 5 mM ATP at 37 °C for 2 h. The reaction mixture (10 μl) was subjected to 10% Tricine SDS-PAGE. (A) Nukacin ISK-1 activity was visualized using an overlay assay, as described in Materials and methods, with Lactobacillus sakei subsp. sakei JCM 1157T as the indicator strain. Lane 1, modified His–NukA; lane 2, modified His–NukA incubated with control membrane; lane 3, modified His–NukA incubated with NukT membrane; and lane 4, 3 μM purified nukacin ISK-1. The arrow indicates the position of nukacin ISK-1. (B) MALDI-TOF mass spectrum after incubation of modified His–NukA with NukT membrane. A peak corresponding to nukacin ISK-1 (2960 Da) is detected. (C) The remaining substrates in the reaction mixture were visualized using western blotting with anti-histidine-tag antibody. Lane 1, modified His–NukA incubated with control membrane; lane 2, modified His– NukA incubated with NukT membrane; lane 3, His–NukA incubated with control membrane; and lane 4, His–NukA incubated with NukT membrane. The black and white arrowheads indicate the position of modified His–NukA and unmodified His–NukA, respectively.
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FIG. 4. Effects of ATP on NukT peptidase activity. Lane 1, 3 μM nukacin ISK-1. Modified His–NukA (3 μM) and NukT membrane (0.5 mg/ml) were incubated with 5 mM ATP (lane 2), without ATP (lane 3), with 5 mM ATP-γ-S (non-hydrolyzable ATP analogue; lane 4), and with 5 mM ATP and 5 mM sodium orthovanadate (ATPase inhibitor; lane 5). Nukacin ISK-1 activity was visualized as in Fig. 2A. The arrow indicates the position of nukacin ISK-1.
were analyzed by western blotting. In contrast to the result obtained using modified His–NukA as a substrate, the signal corresponding to His–NukA remained visible after 2-h reactions with NukT membrane (Fig. 2C, lane 4) and control membrane (Fig. 2C, lane 3); this indicated that NukT does not process His–NukA and that the unusual amino acids in NukA are required for substrate recognition by NukT. Effects of protease inhibitors The similarity between the sequences of NukT and other AMS proteins suggests that the Nterminal domain of NukT is a cysteine protease (6). To characterize NukT-mediated leader peptide cleavage, we determined the effects of the inhibitors of serine/cysteine protease (1 mM PMSF and 5.6 mM E64), aspartic acid protease (14 μM pepstatin), and metalloprotease (3.5 mM phosphoramidon and 5 mM EDTA) on NukT peptidase activity using an in vitro peptidase assay followed by Tricine SDS-PAGE and an overlay assay (described in Materials and methods). PMSF inhibited nukacin ISK-1 production (Fig. 3, lane 2), whereas E-64, pepstatin, and phosphoramidon did not affect NukT peptidase activity (Fig. 3, lanes 3–5). This result supports the sequence prediction that NukT is a cysteine protease. It should be noted that phosphoramidon did not affect NukT peptidase activity, but EDTA did (Fig. 3, lane 6), suggesting that, although NukT is not a metalloprotease, some metal ions may support NukT peptidase activity. Effect of ATP hydrolysis It is generally accepted that the ATPase activity of ABC transporters supplies the energy required for ABC protein-dependent transport. The C-terminal domain of NukT is predicted to be an ATP-binding domain; we examined the effect of ATP hydrolysis on NukT peptidase activity using an in vitro peptidase assay followed by Tricine SDS-PAGE and an overlay assay. Unlike the reaction mixture containing 5 mM ATP, the ATP-free mixture did not exhibit peptidase activity (Fig. 4, lanes 2 and 3). NukT-mediated processing was completely abolished by substituting ATP with 5 mM ATP-γ-s (a non-hydrolyzable ATP analogue) (Fig. 4, lane 4). Additionally, 5 mM vanadate (an ATPase inhibitor) inhibited the NukT activity in the presence of ATP (Fig. 4, lane 5). The unreacted substrates in the reaction mixtures that did not exhibit nukacin ISK-1 activity were detected by western blotting (data not shown). These results suggest that ATP hydrolysis provides sufficient energy for NukT peptidase activity. Furthermore, this activity was not observed in the absence of Mg2+ (data not shown), indicating that Mg2+ is an essential co-factor of NukT. DISCUSSION NukT (a probable AMS protein) is a bifunctional protein that is predicted to mediate leader peptide cleavage and export of type-A(II) lantibiotic nukacin ISK-1. Generally, it is difficult to express membrane proteins from gram-positive bacteria in the gram-negative E. coli; for example, attempts to express full-length ComA (an AMS protein from S. pneumoniae) in E. coli have failed (8). Only the N-terminal peptidase
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domains (ca. 150 amino acids) of some AMS proteins from grampositive bacteria have been expressed in E. coli, and the peptidase activity of these proteins has been characterized (6, 8, 10). Recently, in vitro reconstitution of the peptidase activity of the N-terminal domain of LctT was reported; LctT transports lacticin 481, which is classified under the same type of lantibiotics as nukacin ISK-1 (10). In this study, we successfully expressed full-length NukT in S. carnosus TM300. To characterize the peptidase activity of full-length NukT, we first developed an in vitro assay using inside-out membrane vesicles of S. carnosus expressing NukT. We found that NukT cleaves off the leader peptide of modified His– NukA containing unusual amino acids, but it did not affect leader peptide cleavage of unmodified His–NukA (Fig. 2). This result suggests that the unusual amino acids in NukA are required for substrate recognition by NukT and may also inhibit cleavage of the C-terminal propeptide moiety after recognition by NukT. It has been reported that the first 150 amino acids of LctT can process LctM-treated LctA prepeptide containing three lanthionine rings with similar efficacy to unmodified LctA; this indicates that the rings are not responsible for substrate recognition by the N-terminal domain of LctT (10). On the other hand, the peptidase activity of NisP (a serine protease involved in processing of the leader peptide of type-A(I) lantibiotic nisin) depends on the thioether rings in nisin prepeptide (22). NukT and LctT are AMS proteins involved in the biosynthesis of type-A(II) lantibiotics and exhibit 49% similarity (full-length proteins); their N-terminal peptidase domains show 45% similarity. The substrate specificity of NukT was different from that of the N-terminal domain of LctT but is otherwise poorly understood. At present, we have no data regarding substrate recognition rules for the N-terminal domain of NukT. On the basis of our data, we propose that the substrate recognition mechanism of full-length NukT differs from that of its N-terminal peptidase domain. We found that signals corresponding to the leader peptide were not detected after the in vitro reaction. In contrast, the study of ComA and LctT detected the leader peptides of their respective substrates after the reaction (8, 10). The leader peptide is small and linear and may therefore be easily digested into small fragments by nonspecific proteases present in inside-out membrane vesicles after being processed by NukT or by a specific protease activity of NukT. Sequence comparisons revealed that the Cys12 and His90 of NukT were completely conserved in other cysteine proteases; this suggested that NukT, like other AMS proteins, is a papain-like cysteine protease (6). In this study, the peptidase activity of NukT was completely inhibited by the serine/cysteine protease inhibitor PMSF (Fig. 3), thus strongly supporting the sequence prediction. Surprisingly, E-64, which is also a serine/cysteine protease inhibitor, did not inhibit NukT peptidase activity. Similarly, the N-terminal domain of ComA was not sensitive to E-64 (8). Since AMS proteins exhibit greater substrate specificity than protein-degrading proteases, NukT or the Nterminal domain of ComA might tolerate typical peptide-mimetic serine/cysteine protease inhibitors. One of the interesting findings in this study is that NukT peptidase activity required ATP hydrolysis (Fig. 4). NukT did not cleave off leader peptide without ATP or in the presence of ATP-γ-S (instead of ATP) or vanadate (an ATPase inhibitor). This suggested that ATP hydrolysis provides sufficient energy for NukT peptidase activity. In contrast, the N-terminal peptidase domains of AMS proteins such as LagD, ComA, CvaB, and LctT do not require ATP for their peptidase activity (6, 8–10). The peptidase activity of full-length CvaB (a specific colicin V exporter and an AMS protein produced by gram-negative bacteria) has been characterized. Zhong et al. (23) have suggested that the processing of colicin V precursor by full-length CvaB requires an intact CvaA–CvaB– TolC complex and energy in the form of nucleotide hydrolysis (23). Furthermore, nucleotide binding was required for CvaB dimerization (24). ABC transporters require ATP to couple the hydrolysis of ATP to
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the translocation of solutes across a biological membrane. ATP hydrolysis is coupled to conformational changes in the transporter that mediate the movement of substrate across the membrane (25). Clp, Lon, and FtsH proteases are ATP-dependent proteases that use the free energy of ATP hydrolysis to unfold protein substrates and present them successively to protease active sites. Crystallographic and biochemical studies of these proteins have proposed that the ATPase module unfolds, translocates, and activates proteolytic sites through conformational changes induced by repetitive ATPase cycles (26–28). As one possibility, NukT may utilize the energy of ATP hydrolysis to activate proteolytic sites through conformational changes, and this may determine the substrate specificity of its peptidase domain. Besides ATP, NukT also required Mg2+ for its peptidase activity (data not shown), which explains why EDTA inhibited NukT peptidase activity (Fig. 3, lane 6). Mg2+ is important for ATPase to orient all residues active in catalysis in a productive formation (29); therefore, Mg2+ chelation by EDTA would result in NukT inactivation. While the N-terminal domain of CvaB requires Ca2+ as a co-factor, NukT did not (data not shown). NukT is comparable to the N-terminal domain of ComA or LctT, both of which do not require Ca2+ (8, 10). In summary, full-length NukT cleaves off the leader peptide of modified His–NukA in vitro. NukT may be a cysteine protease; its peptidase activity depends on ATP hydrolysis and the presence of unusual amino acids in NukA. This is the first study to report in detail the peptidase activity of LanT of a type-A(II) lantibiotic. It should be emphasized that our findings provide a novel insight into the molecular basis of lantibiotic biosynthesis and will facilitate the biological production of the engineered lantibiotics. Further studies on full-length NukT are required to provide an understanding of the characteristics of AMS proteins. ACKNOWLEDGMENTS We are grateful to F. Götz (University of Tübingen) for providing S. carnosus TM300 and pTX15. This study was partially supported by grants from the Japan Society for the Promotion of Science (JSPS), Japan Science Society, Novartis Foundation (Japan) for the Promotion of Science, Novozymes Japan Research Fund, and Nagase Science and Technology Foundation. References 1. de Vos, W. M., Kuipers, O. P., van der Meer, J. R., and Siezen, R. J.: Maturation pathway of nisin and other lantibiotics: post-translationally modified antimicrobial peptides exported by gram-positive bacteria, Mol. Microbiol., 17, 427–437 (1995). 2. Sahl, H.-G., Jack, R. W., and Bierbaum, G.: Biosynthesis and biological activities of lantibiotics with unique post-translational modifications, Eur. J. Biochem., 230, 827–853 (1995). 3. McAuliffe, O., Ross, R. P., and Hill, C.: Lantibiotics: structure, biosynthesis and mode of action, FEMS Microbiol. Rev., 25, 285–308 (2001). 4. Chatterjee, C., Paul, M., Xie, L., and van der Donk, W. A.: Biosynthesis and mode of action of lantibiotics, Chem. Rev., 105, 633–684 (2005). 5. Dufour, A., Hindre, T., Haras, D., and Le Pennec, J. P.: The biology of lantibiotics from the lacticin 481 group is coming of age, FEMS Microbiol. Rev., 31, 134–167 (2007). 6. Havarstein, L. S., Diep, D. B., and Nes, I. F.: A family of bacteriocin ABC transporters carry out proteolytic processing of their substrates concomitant with export, Mol. Microbiol., 16, 229–240 (1995). 7. Franke, C. M., Tiemersma, J., Venema, G., and Kok, J.: Membrane topology of the lactococcal bacteriocin ATP-binding cassette transporter protein LcnC. Involvement of LcnC in lactococcin maturation, J. Biol. Chem., 274, 8484–8490 (1999).
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