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Applied and Environmental Microbiology, November 2008, p. 6591-6597, Vol. 74, No. 21
0099-2240/08/$08.00+0     doi:10.1128/AEM.01334-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Mechanistic Dissection of the Enzyme Complexes Involved in Biosynthesis of Lacticin 3147 and Nisin ^,

Anneke Kuipers, Jenny Meijer-Wierenga, Rick Rink, Leon D. Kluskens, and Gert N. Moll^*

BiOMaDe Technology Foundation, Nijenborgh 4, 9747 AG Groningen, The Netherlands

Received 14 June 2008/ Accepted 6 September 2008

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The thioether rings in the lantibiotics lacticin 3147 and nisin are posttranslationally introduced by dehydration of serines and threonines, followed by coupling of these dehydrated residues to cysteines. The prepeptides of the two-component lantibiotic lacticin 3147, LtnA1 and LtnA2, are dehydrated and cyclized by two corresponding bifunctional enzymes, LtnM1 and LtnM2, and are subsequently processed and exported via one bifunctional enzyme, LtnT. In the nisin synthetase complex, the enzymes NisB, NisC, NisT, and NisP dehydrate, cyclize, export, and process prenisin, respectively. Here, we demonstrate that the combination of LtnM2 and LtnT can modify, process, and transport peptides entirely different from LtnA2 and that LtnT can process and transport unmodified LtnA2 and unrelated peptides. Furthermore, we demonstrate a higher extent of NisB-mediated dehydration in the absence of thioether rings. Thioether rings apparently inhibited dehydration, which implies alternating actions of NisB and NisC. Furthermore, certain (but not all) NisC-cyclized peptides were exported with higher efficiency as a result of their conformation. Taken together, these data provide further insight into the applicability of Lactococcus lactis strains containing lantibiotic enzymes for the design and production of modified peptides.

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The lantibiotics nisin and lacticin 3147 are produced by some distinct Lactococcus lactis strains and inhibit a broad range of gram-positive bacterial species. Lantibiotics are peptide antibiotics that contain thioether-bridged amino acids (lanthionines and methyllanthionines) (5, 28, 39). By binding to lipid II, the essential precursor of bacterial cell wall peptidoglycan, they inhibit cell wall synthesis and form hybrid pores in the membrane of the target cell (1-3, 10, 46, 47).

Two main classes of lantibiotics are discerned. In one class (class I), comprising nisin (19), dehydration and cyclization are catalyzed by separate LanB and LanC enzymes. The nisin dehydratase NisB dehydrates serines and threonines of the nisin prepeptide NisA. Subsequently, the nisin cyclase NisC couples the formed dehydroalanine or dehydrobutyrine with cysteines to form lanthionine or methyllanthionine, respectively. The ABC transporter NisT exports modified prenisin out of the cell (16, 31).

In the second class, comprising among others lacticin 481 (32, 48) and the two-component lantibiotics lacticin 3147 (37), dehydration and cyclization are performed by bifunctional LanM enzymes (4). During synthesis of lacticin 3147, the lacticin prepeptides LtnA1 and LtnA2 are each modified by a separate modification enzyme (LtnM1 and LtnM2, respectively) (27). Lacticin 3147 also contains other modifications resulting from conversion of dehydroalanines to D-alanines catalyzed by the enzyme LtnJ (6, 38). The modified peptides Ltn and Ltn_β are processed and transported by LtnT. Both components are essential for the activity of the lantibiotic. Ltn resembles mersacidin in its globular shape, and Ltn_β has some structural similarity to nisin (Fig. 1) (24, 26).

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FIG. 1. Nisin (A) and processed, fully modified Ltnβ peptide (B). In Ltnβ, both threonines included in lanthionine ring A and the one threonine included in methyllanthionine ring B are not dehydrated.

The discovery and development of novel antibiotics is urgent because of the increase in resistance to multiple antibiotics. Lantibiotic mutants with modulated activity have previously been described (35). Furthermore, the application of lantibiotic enzymes can generate biostable thioether-bridged therapeutic peptides, which are resistant against proteolytic degradation (15, 21) (M. Haas, L. D. Kluskens, A. Kuipers, R. Rink, S. A. Nelemans, and G. N. Moll, 14 February 2008, patent application WO 2008/018792A2; L. D. Kluskens, S. A. Nelemans, R. Rink, L. de Vries, A. Meter-Arkema, Y. Wang, T. Walther, A. Kuipers, G. N. Moll, and M. Haas, submitted for publication). Thioether-bridged peptides may also have modulated receptor interaction and extended delivery possibilities.

Several studies indicated that lantibiotic-modifying and -transporting enzymes are organized in multimeric complexes (14, 30, 41). It was demonstrated before that cells containing all lacticin 3147 biosynthesis enzymes, except LtnM1, still produced Ltn_β (27). Here, we studied whether in L. lactis either the combination of LtnM2 and LtnT or LtnT alone is functional and whether peptides that are entirely different from LtnA2 are LtnM2 and LtnT substrates.

Intriguingly, three threonines in the Ltn_β peptide (Fig. 1) that are included in the thioether rings are not dehydrated by LtnM2. In contrast, NisB dehydrates threonines much better than serines and can also successfully generate polydehydrobutyrine (33, 34). In addition, we therefore studied the dehydration of lacticin A2-derived peptides corresponding to these ring-containing sequences by dissected enzyme complexes of the lacticin 3147 and nisin biosynthesis machineries.

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Abbreviations.
The abbreviations used in this paper are defined as follows: Dha, dehydroalanine; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; TFA, trifluoroacetic acid; TCEP, Tris(2-carboxyethyl)phosphine; CDAP, 1-cyano-4-dimethylamino-pyridinium tetrafluoroborate; Cm^r, chloramphenicol resistant; and Em^r, erythromycin resistant.

Bacterial strains and plasmids.
L. lactis NZ9000 was used for expression of the modification enzymes and peptides. The modification genes and transporter genes ltnM2-ltnT and nisBTC were cloned into pIL253-derived plasmids (42) behind the nisin-inducible promoter. The sequences encoding the N-terminal lacticin leader LtnA2 or the nisin leader were fused to the sequence encoding the substrate peptide and placed under the control of the nisin-inducible promoter of pNZ8084-derived plasmids (20). The strains and plasmids are listed in Table 1.

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TABLE 1. Bacterial strains and plasmids

Molecular cloning.
The ltnA2 and ltnT-ltnM2 genes were amplified from pBAC105, isolated from L. lactis IFPL105, and the nisA and nisBTC genes were amplified from chromosomal DNA of L lactis NZ9700. PCRs were performed with Phusion DNA polymerase (Finnzymes, Finland). Restriction enzymes used for cloning strategies were purchased from New England Biolabs, Inc. Ligation was carried out with T4 DNA ligase (Roche, Mannheim, Germany). Sequences encoding peptides different from the original propeptides were genetically fused to the leader or propeptide by means of PCR (18). Electrotransformation of L. lactis was carried out as previously described (12), using a Bio-Rad gene pulser (Richmond, CA). Nucleotide sequence analysis was performed by BaseClear (Leiden, The Netherlands).

Growth conditions.
L. lactis was grown in M17 broth (43) supplemented with 0.5% glucose (GM17) or in minimal medium (13, 33) with or without chloramphenicol (5 µg/ml) and/or erythromycin (5 µg/ml). Cultures were grown on minimal medium after induction with nisin prior to sample preparation for mass spectrometry.

Mass spectrometry.
Samples were purified from the medium fraction by zip tip purification (C₁₈ zip tip; Millipore) or directly spotted by putting 1 µl of the supernatant on the target. After drying, the spots were washed once with 4 µl Milli-Q water to remove the salts. Subsequently, 1 µl of a matrix (10 mg/ml -cyano-4-hydroxycinnamic acid in 50% acetonitrile containing 0.1% [vol/vol] TFA) was added to the target and allowed to dry. CDAP was used to react with free cysteine residues. The vacuum-dried sample was resuspended in 9 µl 25 mM citrate buffer, pH 3.0, and reduced with 1 µl TCEP (10 mg/ml in MQ). After a 10-min incubation at room temperature, 2 µl of CDAP (10 mg/ml in 100% acetonitrile) was added, followed by 15 min of incubation at room temperature. Mass spectra were recorded with a Voyager DE PRO MALDI-TOF mass spectrometer in the linear mode (Applied Biosystems). In order to maintain high sensitivity, an external calibration was applied. All measurements were performed in at least three independent experiments, with identical results.

Gel electrophoresis.
L. lactis cells were induced with nisin and grown further in minimal medium. Peptides were isolated from the supernatant by using disposable solid phase extraction Bond Elut C₁₈ cartridges from Varian. After the supernatant was applied, the column was washed with MQ containing 0.1% TFA and peptides were eluted with 100% methanol containing 0.1% TFA. Peptides from 1 ml of supernatant were applied on Tricine gel (40). Analysis was performed using a silver staining kit (Invitrogen) or by Coomassie staining.

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Functionality of the enzymes LtnM2 and LtnT.
In order to analyze the functionality of the enzymes LtnM2 and LtnT in L. lactis, pILPTM2 was coexpressed with pA24, encoding the natural substrate LtnA2. MALDI-TOF analysis of the supernatant revealed a mass of 2,846 Da, identical to the expected mass of the eightfold-dehydrated Ltn_β peptide, which lacks conversions of two dehydroalanines to D-alanines due to the absence of the enzyme LtnJ (6, 38) and has undergone an N-terminal deamination yielding an -keto amide (+1 Da) (24) (see Fig. S1 in the supplemental material). Clearly, the enzymes LtnM2 and LtnT, in the absence of any other lacticin 3147 enzyme, successfully modified, processed, and translocated the prepeptide LtnA2.

LtnM2-LtnT dehydration, processing, and export peptide comprising angiotensin-(1-7) variants.
We assessed the versatility of LtnM2-LtnT for modifying and producing angiotensin-(1-7) variants and compared the results with those for an existing angiotensin-producing NisBTC system. Sequences coding for peptides comprising nisin positions 1 to 17 and the angiotensin-(1-7) DKTYICP, DATYICP, and DTVYCHP variants were placed behind the lacticin A2 leader or the nisin leader. The encoding plasmids were each coexpressed in L. lactis with pILPTM2 or pIL3BTC, respectively. The extent of LtnM2-LtnT- or NisBTC-mediated dehydration and translocation of the peptides in the supernatant of induced cultures was analyzed by MALDI-TOF mass spectrometry (Table 2 and Fig. 2A and B; also see Fig. S2A and B in the supplemental material) and silver-stained Tricine gel (Fig. 3). The amount of peptide produced via the NisBTC system (Fig. 3, lanes 4 to 6) was higher than that produced via the LtnM2-LtnT system (Fig. 3, lanes 1 to 3). LtnM2-LtnT-exported, -dehydrated, and -processed peptides were clearly detectable by MALDI-TOF mass spectrometry (Fig. 2B; also see Fig. S2A and B in the supplemental material). All three peptides were evidently dehydrated up to fivefold by NisB (Table 2 and Fig. 2A), whereas LtnM2-mediated dehydration was in two cases up to fivefold and for the peptide encoded by pA25-7.8 even up to sixfold (fully) (Table 2 and Fig. 2B; also see Fig. S2A and B in the supplemental material). In all cases, fivefold dehydration was the major product, implying that the efficiencies of LtnM2- and NisB-mediated dehydration of the peptides did not differ to a large extent.

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TABLE 2. Comparison of peptide sequences fused to the nisin leader, modified and secreted by NisBTC with the same peptide sequences fused to the LtnA2 leader, modified, processed, and secreted by LtnM2-LtnT

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FIG. 2. Dehydration of angiotensin fusion peptides by NisB and LtnM2. Culture supernatant was analyzed by MALDI-TOF mass spectrometry as described in Materials and Methods. (A) Supernatant of L. lactis NZ9000 (pIL3BTC, pNZang1-7.8). The expected mass of the protonated and fully dehydrated STKDFNLDLVSVSKKDSGASPRITSISLCTPGCKTGALMIEGRDTVYCHP fusion peptide is 5,194 Da. The main peak, 5,212 Da, represents the fivefold-dehydrated peptide. (B) Supernatant of L. lactis NZ9000(pILPTM2, pA25-7.8). The expected mass of the processed, protonated, and fully dehydrated ITSISLCTPGCKTGALMIEGRDTVYCHP peptide is 2,861 Da.

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FIG. 3. Secretions of modified peptides by LtnT and by NisT. L. lactis cells were induced with nisin and grown overnight in minimal medium. Peptides from 1 ml of supernatant were applied on a Tricine gel. The gel was stained using a silver staining kit (Invitrogen). Lanes: 1, NZ9000(pILPTM2, pA25-7.2); 2, NZ9000(pILPTM2, pA25-7.3); 3, NZ9000(pILPTM2, pA25-7.8); 4, NZ9000(pIL3BTC, pNZang1-7.2); 5, NZ9000(pIL3BTC, pNZang1-7.3); 6, NZ9000(pIL3BTC, pNZang1-7.8); 7, kaleidoscopic marker (Bio-Rad). In lanes 4, 5, and 6, dimers were clearly detected, likely resulting from high peptide concentrations.

The observed masses of the peptides in the supernatant indicated correct LtnT-mediated processing and export. In the absence of the transporter, no peptide was detected in the supernatant, excluding lysis and other translocation mechanisms. These data clearly prove that LtnM2 is capable of dehydrating peptides that are entirely different from its natural substrate. Importantly, these data also show that LtnT can subsequently remove the leader peptide and translocate the modified peptides in vivo.

LtnT stays active in the absence of LtnM2.
We investigated whether LtnT itself, in the absence of the other lacticin 3147 synthetase enzymes, retains activity. When LtnT was coexpressed with LtnA2 or the DTVYCHP-comprising peptide preceded by the LtnA2 leader, processed peptides were identified in the supernatant (see Fig. S3A and B in the supplemental material). In the absence of the transporter, no peptide was detected in the supernatant, excluding lysis and other translocation mechanisms. These results indicate that functional dissection of LtnT from the other synthetase enzymes is possible. LtnT can process and translocate LtnA2 and unrelated substrate peptides, provided that the LtnA2 leader is present.

Do thioether rings prevent the dehydration of threonines in the peptide Ltn_β?
In the natural, posttranslationally modified Ltn_β peptide, two threonines included in the first lanthionine ring and one threonine included in the second methyllanthionine are not dehydrated by LtnM2 (Fig. 1). In order to determine whether dehydration is impaired by the thioether rings installed by LtnM2, an LtnM2 mutant in which the cyclase activity was knocked out by exchanging the conserved cysteine at position 818 in LtnM2 was made (23), resulting in plasmid pILPTM2-C818A. However, when LtnA2-encoding pA24 was coexpressed with pILPTM2-C818A, secretion of the dehydrated natural substrate LtnA2 was not detected in the supernatant by MALDI-TOF mass spectrometry. In the absence of cyclase activity, spontaneous formation of nonoriginal large rings in the LtnA2 peptide cannot be excluded, and these might hamper processing or transport by LtnT. The peptide variants comprising angiotensin-(1-7), encoded by plasmids pA25-7.2 and pA25-7.8 and coexpressed with pILPTM2-C818A, were successfully dehydrated, processed, and translocated, indicating that the dehydratase activity of C818A-LtnM2 and the processing and transport activity of LtnT were unaltered (data not shown).

Moreover, when the TTPATPAISILSAYI (pA27) and ILSAYISTNTCPTTKCTRAC (pA28) LtnA2 peptide truncations were preceded by the lacticin A2 leader peptide and coexpressed with the enzymes LtnM2 and LtnT, no secreted peptides were detected, either processed or unprocessed. Apparently, processing and/or transport by LtnM2-LtnT has substrate-specific limitations. For further studies of the possible influence of thioether rings on dehydration, we therefore continued this study by applying the nisin biosynthesis NisBT or NisBTC enzymes.

Enhanced dehydration in the absence of NisC.
When the truncated ILSAYISTNTCPK and ILPTTKCTRAC LtnA2 peptide variants, preceded by the nisin leader and encoded by plasmids pNZ29 and pNZ30, were coexpressed with pIL3BT (Fig. 4A and C) or pIL3BTC (Fig. 4B and D), a higher extent of dehydration was observed in the absence of NisC. In this context, it seems that the threonine residues that are not dehydrated by LtnM2 can be dehydrated by NisB. To verify whether unmodified threonines in the thioether rings were involved, a C-terminally adapted fragment of LtnA2, ILPTTKCARAA, was fused to the nisin leader (pNZ31). Coexpression of pNZ31 with pIL3BT (Fig. 4E) or pIL3BTC (Fig. 4F) showed again a higher extent of dehydration of the peptide in the absence of NisC. Incubations with TCEP and CDAP and the lack of CDAP modification confirmed that the ILPTTKCARAA peptide was almost fully cyclized by NisC (see Fig. S4A and B in the supplemental material).

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FIG. 4. Enhanced dehydration by NisBT compared to that by NisBTC. Culture supernatant was analyzed by MALDI-TOF mass spectrometry. The expected masses of the fully dehydrated and protonated fusion peptides are as follows: for that encoded by pNZ29 (nisin leader fused to ILSAYISTNTCPK), 3,672 Da; for that encoded by pNZ30 (nisin leader fused to ILPTTKCTRAC), 3,487 Da; and for that encoded by pNZ31 (nisin leader fused to ILPTTKCARAA), 3,443 Da. (A) pNZ29 coexpressed with pIL3BT; the main peak is for a peptide fourfold dehydrated. (B) pNZ29 coexpressed with pIL3BTC; the main peak is for a peptide twofold dehydrated. (C) pNZ30 coexpressed with pIL3BT; the main peak is for a peptide twofold dehydrated. (D) pNZ30 coexpressed with pIL3BTC; the main peak is for a peptide onefold dehydrated. (E) pNZ31 coexpressed with pIL3BT. (F) pNZ31 coexpressed with pIL3BTC.

Control experiments provided consistent data. The cysteines in the ILPTTKCTRAC peptide were exchanged for alanines, leading to ILPTTKARAA, encoded by pNZ32. This plasmid was coexpressed with pIL3BTC (see Fig. S5A in the supplemental material) and pIL3BT (see Fig. S5B in the supplemental material), which resulted in identical extents of dehydration. Furthermore, knocking out the cyclase activity by the mutation C326A (23), resulting in the plasmid pIL31BTdC, did not alter the extent of peptide dehydration. Identical dehydration levels were observed upon coexpression with pIL31BTdC and pIL3BT (data not shown). Expression of the activity-deficient NisC mutant was confirmed by Western blotting (data not shown). All these data clearly demonstrate reduced dehydration in the presence of the cyclase NisC, consistent with reduced dehydration of threonines that are included in the thioether rings.

NisC-mediated cyclization enhances the production levels of some peptides but not those of others.
We investigated whether or not the presence of NisC might enhance export. The amounts of secreted peptide produced via NisBT and NisBTC were compared and analyzed with a Coomassie-stained Tricine gel (Fig. 5). No noteworthy difference in secretion level was observed between NisBT- and NisBTC-transported peptides when peptide-encoding plasmid pNZ29 was coexpressed with pIL3BT or pIL31BTC. The secretion level of the peptide encoded by pNZ30 was somewhat higher upon coexpression with pIL3BTC instead of pIL3BT. However, when pNZ31 was coexpressed with pIL3BTC, the secretion level was clearly higher than when pNZ31 was coexpressed with pIL3BT.

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FIG. 5. Secretion of peptides via NisBT and NisBTC. L. lactis cells were induced at an optical density at 600 nm of 0.4 with nisin and grown further in minimal medium for 5 h. Peptides from 3 ml of supernatant were applied on a Tricine gel and analyzed by Coomassie staining. Lanes: A, kaleidoscopic marker (Bio-Rad); 1, NZ9000(pIL3BT, pNZ29) (nisin leader fused to ILSAYISTNTCPK); 2, NZ9000(pIL3BTC, pNZ29); 3, NZ9000(pIL3BT, pNZ30) (nisin leader fused to ILPTTKCTRAC); 4, NZ9000(pIL3BTC, pNZ30); 5, NZ9000(pIL3BT, pNZ31) (nisin leader fused to ILPTTKCARAA); 6, NZ9000(pIL3BTC, pNZ31).

Subsequently, we wanted to distinguish a channeling effect of NisC itself from enhanced export of peptides due to the cyclized peptide conformation. When peptides without cysteines, hence without potential for thioether ring formation, such as the ones encoded by pNZ31 (pTP-DSRWARVALIDSQKAAVDKAITDIAEKL) and pTPtppii (see Fig. S6 in the supplemental material), were coexpressed with either pIL3BT or pIL3BTC, no differences in transport were detected. Nor did the presence of the inactive NisC mutant C326A improve transport efficiency (data not shown). These observations indicate that in these experiments no channeling effect of NisC for transport of peptides by NisT occurs. Hence, differences between export via NisBT and that via NisBTC, observed for the peptides encoded by plasmids pNZ29 and pNZ30, are likely caused by differences in conformation between (methyl)lanthionine-containing peptides and linear peptides with free cysteines.

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We here studied posttranslational modifications, processing, and transport by (sub-) complexes of class I and class II lantibiotic enzymes. Since neither the activity of lacticin 3147 M1 and M2 enzymes nor the nisin dehydratase activity of NisB has been reconstituted in vitro, we here performed in vivo studies of the dissection of the enzyme complexes of these two lantibiotics, lacticin 3147 and nisin.

It has been reported that mutagenized LtnA1 and LtnA2 are each still modified, processed, and transported and retain antimicrobial activity (7-9). In all cases, the mutagenized substrates were still highly homologous to their natural counterparts. In the study by Cotter et al. (7), not all of the alanine-substituted peptides were detected. It is not clear whether the lack of detection of some of the mutagenized peptides is due to impaired processing and/or transport.

LtnT is an ABC transporter with a dual function. First, the ABC transporter is a maturation protease; its intracellular proteolytic domain resides in the N-terminal part of the protein. Second, after removal of the leader peptide, LtnT translocates the substrate across the membrane (11). In the nisin biosynthesis machinery, these processes are carried out by two distinct enzymes. NisT transports prenisin across the membrane, and NisP extracellularly removes the leader peptide (45, 49). Hence, removal of the nisin leader is not a prerequisite for transport of peptides fused to the leader (17).

As can be seen in Table S1 in the supplemental material, there is peptide-dependent variation in processing and export. Modification of the peptide substrates does not seem to be a qualification for processing and transport by LtnT. Clearly, more research is needed to establish a clear picture of the substrate specificity of the lacticin 3147 synthetase enzymes and transporter involved.

In this study, we demonstrated for the first time that LtnM2 can dehydrate peptides unrelated to LtnA2 and that LtnT was surprisingly able to process and translocate these modified peptides. Moreover, LtnT was shown to process and transport substrate peptides in the absence of all other lacticin 3147 synthetase enzymes. Xie and coworkers demonstrated in vitro activity of lacticin 481, whose modification enzyme LctM has homology with LtnM2, independent of LctT (48). An in vivo study showed that in the absence of LctT a fully modified but truncated variant of lacticin 481 was detected in the supernatant by mass spectrometry (44). This truncated, modified variant seems to be translocated via another transporter. In addition, NukM can modify its substrate in the absence of NukT (30). The latter study also proposed a membrane-located multimeric enzyme complex in which NukT and NukM are associated like the enzymes of the proposed membrane-associated multimeric lantibiotic synthesis complex (NisBTC/SpaBTC) (14, 41). We here demonstrate that, like the enzymes NisB, NisC, NisT, and NisP, which can function independently (17, 18, 22), the combination of LtnM with LtnT and LtnT alone can also function independent of other lacticin 3147 synthetase enzymes.

We hypothesized that dehydration and cyclization by the enzymes LanM, LanB, and LanC are alternating activities and that thioether rings may inhibit dehydration of residues that are included in the thioether ring. In a previous study by Miller et al., the possibility that cyclization prevented dehydration of specific residues of the lacticin 481 peptide was not excluded (29). For the lacticin 3147 LtnM2 enzyme, we have no conclusive data, as a result of apparent substrate limitations of LtnT. We therefore compared modifications by partially dissected enzyme complexes (NisBT versus NisBTC). All the tested truncations of the LtnA2 peptide had higher extents of dehydration in the absence of NisC. In the case of export via NisBT, fully dehydrated peptides were observed. The apparent impaired dehydration of residues included in the ring is fully consistent with the possibility that the rings themselves interfere with the accessibility of NisB. This points to the occurrence of alternating dehydration and cyclization activities. For alternating activities, it is likely that association between NisB and NisC is needed, consistent with the existence of a multimeric enzyme complex (41). It is tempting to speculate on a model in which the nisin leader might bind close to an interface between NisB and NisC, allowing the modifiable propeptide substrate to flip from NisB to NisC and back again during alternating activities. Although no data are available yet, such a model is even easier to envisage for LctM enzymes in which the dehydratase and cyclase catalytic sites are present within one and the same enzyme.

Lantibiotic cyclases regiospecifically cyclize thioether rings (36, 50). Because of their high reactivity, dehydroalanines may spontaneously react with cysteines. As a result, regiospecific cyclase activity might be required before unwanted thioether bridges are formed. Therefore, alternating activities of dehydratase and cyclase might in some peptides be crucial to ascertain the correct bridging pattern, essential for activity. To provide a complete picture, it should be emphasized that alternating dehydratase and cyclase activities may occur but are not an absolute necessity. For instance, in vitro reconstitution demonstrated that NisC can cyclize fully dehydrated prenisin in the absence of NisB and NisT (22). In these studies, dehydration and cyclization were uncoupled processes and could therefore not be alternating processes. Many lantibiotics possess dehydrated residues in a thioether ring, like Dha5 and Abu25 in nisin. Clearly, while we do not know whether dehydration of these particular residues takes place before or after ring formation, they are dehydrated. Future studies might investigate the possibility that the conformation of the peptide substrate itself affects whether, when and where the dehydration and cyclization activities alternate.

 
L. D. Kluskens is supported by the Dutch Technology Foundation, STW project 06927.

Kathleen Wood is gratefully acknowledged for reading the manuscript.

 
* Corresponding author. Mailing address: BiOMaDe Technology Foundation, Nijenborgh 4, 9747 AG Groningen, The Netherlands. Phone: 31 50 3638070. Fax: 31 50 3634429. E-mail: Moll{at}biomade.nl

Published ahead of print on 12 September 2008.

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

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Applied and Environmental Microbiology, November 2008, p. 6591-6597, Vol. 74, No. 21
0099-2240/08/$08.00+0     doi:10.1128/AEM.01334-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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