Lantibiotics, Biosynthesis and Mode of Action of
L.A. Furgerson-ihnken and Wilfred A. van der Donk, University of Illinois at Urbana-Champaign, Illinois
Lantibiotics are a unique class of antimicrobial peptides produced by Gram-positive bacteria. They are synthesized ribosomally as precursor peptides and undergo extensive posttranslational modification to attain their biologically active structures. These modifications include dehydration of serine and threonine residues that produce dehydroalanines and dehydrobutyrines, followed by intramolecular addition of cysteine thiols to the unsaturated amino acids. The lantibiotic biosynthetic machinery displays low substrate specificity both in vivo and in vitro, which allows for the engineering of novel lantibiotics. The polycyclic peptides exert their biological activity through several modes of action including the sequestration of the cell wall precursor lipid II and pore formation in bacterial membranes. Because of their high potency, several commercial applications of lantibiotics are being developed.
Lantibiotics belong to the bacteriocin family of antimicrobial agents. Although originally identified as antibiotics, the functional diversity of the lantibiotics is much broader than imagined previously. They are posttranslationally modified polycyclic peptides, and its best-known member, nisin, has been employed by the food industry as a preservative for more than 40 years in over 80 countries. Remarkably, no widespread bacterial resistance has developed during this prolonged use. Therefore, lantibiotics represent potentially a new line of defense in the battle against drug-resistant bacteria.
The prototypical lantibiotic, nisin, was discovered in 1928 for its antibacterial properties and has been used as a preservative in dairy products since the 1950s (1). Nisin and other lantibiotics exhibit nanomolar efficacy against many Gram-positive strains of bacteria (2), which include methicillin resistant Staphylococcus aureus, vancomycin resistant enterococci, and oxacillin resistant bacteria. On the other hand, some lantibiotics function as morphogenetic peptides rather than antibiotics and are important for spore formation in streptomycetes (3). Since the structural elucidation of nisin in the early 1970s, extensive research efforts have been directed at understanding the biosynthesis and mode of action of various lantibiotics.
Lantibiotics are synthesized ribosomally as precursor peptides with an amino-terminal leader sequence and a carboxy-terminal structural region. The structural region undergoes extensive posttranslational modification catalyzed by enzymes unique to lantibiotic biosynthesis. After structural region modification, the unaltered leader sequence is removed by a protease, which results in the biologically active species.
Lantibiotics contain several unusual amino acids, including the thioether lanthionine (Lan) linkage and its methyl substituted analog methyllanthionine (MeLan) (Fig. 1a) that unifies all members of the class and accounts for their family name. In addition to Lan, lantibiotics commonly contain 2,3-dehydroalanine (Dha) and (Z)-2,3-dehydrobutyrine (Dhb). In all, no less than 15 different posttranslational modifications have been documented in lantibiotics (for a selection see Fig. 1a), and up to 58% of their amino acids are modified. These extensive structural alterations overcome the constraints imposed by the use of 20 amino acids in ribosomally synthesized peptides. Some less common, posttranslationally crafted residues in lantibiotics are fi-hydroxy aspartate, lysinoalanine, aminovinyl cysteine (AviCys), D-alanine, 2-oxobutyrate, 2-oxopropionate, and 2-hydroxypropionate. The presence of these unusual residues is thought to be important for the biological activity of lantibiotics.
Currently, the lantibiotic family contains more than 50 members with varying structures, properties, and biological activities (Fig. 1b). Depending on the ring topology of mature lantibiotics, they may have an elongated three-dimensional structure (e.g., nisin), a globular conformation (e.g., cinnamycin), or have both regions of linearity and of globular nature (e.g., lacticin 481). The thioether linkages are thought to confer stability and enhance activity by locking the mature lantibiotics in their biologically active form and by decreasing the susceptibility to protease-mediated degradation. The mature species vary widely in size, shape, charge, and biological activity. They have been classified by groupings named after a representative member (e.g., the nisin, lacticin 481, and cinnamycin subgroups) (4). Members of each group share a high degree of sequence homology, biosynthetic machinery, and also have similar biological activities. A unique group of lantibiotics is the two-component systems such as lacticin 3147 (Fig. 1b) that are composed of two posttranslationally modified peptides that act in synergy to exert their bactericidal activity.
Figure 1. (a) Selection of the structural motifs found in lantibiotics that result from posttranslational modification. A shorthand notation for each structure is also illustrated. (b) Representative structures of mature lantibiotics using the shorthand notation.
Mode of action
Nisin displays several biological activities including inhibition of cell wall biosynthesis, disruption of the cell membrane, and inhibition of spore outgrowth. Nisin is able to disrupt cell wall biosynthesis by binding to the essential peptidoglycan precursor lipid II (Fig. 2a) (2). Based on studies with fluorescently labeled nisin, the molecule not only binds lipid II, but also removes it from its functional location in the cell (7). Mutagenesis studies have demonstrated that the A and B rings of nisin are essential for binding to lipid II (5), and a nuclear magnetic resonance (NMR) structure of a 1:1 nisin-lipid II analog complex has revealed that the mechanism of molecular recognition involves hydrogen bonding interactions between the lipid II pyrophosphate moiety and the nisin amide linkages in the A and B-rings (Fig. 2b) (8). After binding to lipid II at the membrane surface, the C-terminus of nisin inserts perpendicularly into the membrane (Fig. 2c). Mutagenesis studies have also shown that the C and D rings of nisin as well as a hinge region are important for pore formation (8). This hinge region, which is composed of three residues that link the C and D rings (Asn20, Met21, and Lys22), gives nisin the conformational flexibility necessary to traverse the lipid bilayer. The mechanism of bacterial spore outgrowth inhibition is less defined, but it is thought to involve the electrophilic nature of Dha5 (9).
The mode of action of the two-component lantibiotic lacticin 3147 also involves binding to lipid II and pore formation, but it requires two posttranslationally modified peptides to act in synergy to affect their bactericidal properties. In a recent model, lacticin 3147 A1 was proposed to first bind lipid II, which causes a change in its conformation such that lacticin 3147 A2 is recruited to the cell surface. Interaction of A2 then promotes deeper insertion into the membrane and pore formation (10).
Other lantibiotics, like mersacidin, also bind lipid II but do not form pores. They are thought to prevent the transglycosylation step of cell wall biosynthesis by preventing action of the transglycolase enzyme on lipid II. This inhibition is achieved by binding to the disaccharide pyrophosphate region of the peptidoglycan precursor (11). Vancomycin also prevents cell wall biosynthesis by binding the D-Ala-D-Ala moiety of lipid II. Considering their different binding sites, it is not surprising that alteration of the D-Ala-D-Ala structure in vancomycin resistant bacterial strains does not affect the efficacy of mersacidin (11). Thus, mersacidin represents a promising antibiotic candidate with the potential of treating multi-drug resistant bacterial infections.
The cinnamycin group of lantibiotics inhibits the activity of phospholipase A2 by binding to phosphatidylethanolamine (12), which affects prostaglandin and leukotriene biosynthesis by preventing the release of arachidonic acid from the cell membrane. Furthermore, the duramycins, also part of the cinnamycin group, promote chloride secretion in airway epithelia and are evaluated for cystic fibrosis treatment (13). The most recent new activity of lantibiotics was discovered in the peptides SapB and SapT produced by Streptomyces coelicolor and S. tendae, respectively (3). These peptides are capable of self-assembly at air-water interfaces and are important for the formation of nascent aerial filaments during sporulation.
Figure 2. Mode of action of the prototypical lantibiotic nisin. (a) The peptidoglycan precursor lipid II is composed of an N-acetylglucosamine-β-1,4-N-acetylmuramic acid disaccharide (GlcNAc-MurNAc) that is attached to a membrane anchor of 11 isoprene units via a pyrophosphate moiety. A pentapeptide is linked to the muramic acid. Transglycosylase and transpeptidase enzymes polymerize multiple lipid II molecules and crosslink their pentapeptide groups, respectively, to generate the peptidoglycan. (b) The NMR solution structure of the 1:1 complex of nisin and a lipid II derivative in DMSO (6). (c) The amino-terminus of nisin binds the pyrophosphate of lipid II, whereas the carboxy-terminus inserts into the bacterial membrane. Four lipid II and eight nisin molecules compose a stable pore, although the arrangement of the molecules within each pore is unknown (5).
The genes that encode lantibiotics and their biosynthetic machinery are designated generically by the locus symbol lan, with a more specific annotation for each family member. The lanA gene encodes the precursor peptide LanA, with NisA used for the nisin precursor, LctA for the lacticin 481 prepeptide, and so on. The lanBCDMPTXY genes encode for various modification enzymes, the lanKRQX genes produce regulatory proteins, and the lanFEGI genes provide immunity proteins. Typically, a subset of these genes is present in a given biosynthetic gene cluster. The cluster for nisin is shown in Fig. 3a as a representative example.
Enzymatic dehydration and cyclization
The unsaturated residues Dha and Dhb are formed by dehydration of serine and threonine residues, respectively, and the thioether linkages Lan and MeLan are generated by intramolecular Michael-type addition of cysteine thiols to the unsaturated sites (e.g., Fig. 3b). These modifications can be performed by either two separate enzymes (LanB and LanC) in class I lantibiotics or a single bifunctional enzyme (LanM) in class II lantibiotics. Typically, proteolysis of the leader sequence is performed by a dedicated protease, either a LanP serine protease (class I) or the cysteine protease domain of a LanT protein (class II). The lanB genes encode large (~1000 residues) predominantly hydrophilic dehydratases that may be membrane associated. To date, the dehydratase activity of a LanB protein has not been reconstituted in vitro and little is known about the mechanism of catalysis of this group of enzymes.
The lanC genes encode smaller (~400 residues), zinc metalloproteins that catalyze cysteine thiol addition to unsaturated amino acids to form the lanthionine and methyllanthione residues in a regioselective, stereoselective, and chemoselective manner (Fig. 3b). The first experimental evidence for the role of LanC proteins in lantibiotic biosynthesis was the accumulation of dehydrated NisA in strains that lack the NisC protein (14). In 2006, the crystal structure of NisC was solved, and its cyclase activity was reconstituted in vitro (15). Its active site contains a single zinc atom with one histidine and two cysteine ligands as well as a coordinated water molecule. A cysteine thiol of the substrate is thought to displace this water molecule, followed by deprotonation to generate a thiolate that then attacks the β-carbon of a Dha or Dhb residue, which results in an enolate that is protonated stereoselectively to provide the D-configuration at the α-carbon (Fig. 3c).
Class II lantibiotic gene clusters (e.g., lacticin 481) do not contain lanBC genes, but instead contain a lanM gene that encodes a large bifunctional LanM protein (~115-120 kDa) that catalyzes both dehydration and cyclization (Fig. 4). The C-termini of LanM proteins share a low degree of sequence identity with LanC proteins, which include the zinc binding ligands, but have no homology to LanB proteins. Both the dehydratase and cyclase activities of lacticin 481 synthetase (LctM) and haloduracin synthetase (HalM) were reconstituted recently in vitro (16, 17).
Figure 3. (a) The nisin biosynthetic gene cluster. (b) Posttranslational modifications during the biosynthesis of nisin. Dehydration of serine and threonine residues in the structural region of the precursor peptide NisA is performed by the dehydratase NisB. Then, the (Me)Lan rings are installed by the cyclase NisC. After secretion, the unmodified leader sequence is removed by the serine protease NisP, which generates the biologically active species. (c) The proposed cyclization mechanism for NisC.
Another common posttranslational modification found in mature lantibiotics is a C-terminal AviCys (Fig. 1a). This residue is formed by EpiD in epidermin (18). Currently, not much is known about the enzymes that catalyze formation of other unique lantibiotic residues. The cinnamycin gene cluster was cloned recently (19), which set the stage for studies to determine which gene products are responsible for lysinoalanine and β-hydroxy aspartate formation.
Proteolysis and export
After posttranslational modification of the precursor peptide, the unmodified leader sequence is cleaved by a protease to reveal the biologically active compound. Depending on the lantibiotic, this cleavage occurs either just prior to or after transport outside the cell. For most class I lantibiotics, the cleavage reaction is catalyzed by a LanP serine-type protease. Many LanP proteases contain a preprosequence, which indicates they themselves may be secreted and therefore act after the modified precursor peptide has been exported. NisP is required for nisin maturation because strains that lack the nisP gene but contain all other nisin biosynthetic machinery produce fully modified but biologically inactive nisin with the leader peptide still attached (20). For all lantibiotics investigated to date, removal of the leader sequence is required for biological activity.
Lantibiotics are secreted out of the cell by the LanT ATP-binding cassette transporters. These enzymes are transmembrane, homodimeric proteins that use the energy of ATP hydrolysis to secrete either the mature lantibiotic or a fully modified precursor peptide with the leader sequence still attached. A second type of LanT transporter is found in the biosynthetic machinery of class II lantibiotics, whose precursor peptides contain a “double-glycine” type cleavage site (21). These bifunctional LanT proteins contain an N-terminal proteolytic domain in addition to the membrane-spanning portion and C-terminal ATP-binding cassette. Cleavage occurs C-terminal to the G(-2)G/A(-1) motif at the junction between leader and structural region, and it is thought to take place concomitant with export.
Chemical Tools and Techniques
Because lantibiotics are gene-encoded antimicrobials, genetic, molecular biology, and protein chemistry techniques have been used traditionally to explore their structure and biosynthesis. These approaches include gene deletions and disruptions, and in vivo and in vitro site-directed mutagenesis. The following sections will detail more recent tools used in various aspects of lantibiotic research with an emphasis on chemical biology techniques.
Until recently, the discovery of new lantibiotics relied on the painstaking isolation and purification of compounds that exhibited bactericidal activity from the producer strains. Characterization of those isolates by amino acid analysis, and in some cases determination of their structure by NMR spectroscopy would classify them as a lantibiotic. More recently, the advent of bioinformatics has provided another avenue to discover new lantibiotics by scanning the ever-increasing number of fully sequenced genomes for genes homologous to known lantibiotic producing genes. The two-component lantibiotic haloduracin was discovered in this way (17, 22).
Over the years, several techniques have been developed to elucidate the structure of a new lantibiotic. The extensive posttranslational modifications limit Edman degradation to a stretch of amino acids from the N-terminus to the first modification. Various chemical derivatization techniques have been used to allow continuation of the sequence and to reveal the position of the posttranslationally modified residues. Originally, these techniques relied on treatment with ethanethiol under highly basic conditions that result in elimination reactions of the thioethers and addition of ethanethiol to both the resulting dehydroamino acids and the dehydrated residues already present in the parent structure. More recently, the structural determination of the LtnA1 and LtnA2 peptides of lacticin 3147 was aided by a novel method that involves nickel boride (Ni2B), an in situ generated hydrogenation and desulfurization catalyst (23). First, the N-terminal 2-oxobutyryl residue and the Dhb at position 2 of LtnA2 (see Fig. 1b) were removed by treatment with 1,2-diaminobenzene in aqueous acetic acid. Subsequent treatment with Ni2B in the presence of NaBD4 in CD3OD/D2O resulted in the incorporation of a single deuterium at the β-carbon of each residue that was involved in a (methyl)lanthionine linkage. Deuterium atoms are introduced at both the a- and β-carbon of dehydro amino acids, which allows distinction between (Me)Lan and Dha/Dhb that was not possible with the ethanethiol method. Both approaches are unable to determine the thioether ring topology, however, and modern NMR techniques and in some select cases tandem mass spectrometry have been used for this task. It should be noted that the thioether linkages restrict the use of tandem mass spectrometry methods to linear stretches of amino acids within the lantibiotics, because fragmentation within a ring does not result in fragment peptides (17). However, when tandem MS techniques are used in combination with site-directed mutagenesis to remove one or more rings from the parent structure, it offers a highly valuable approach to establish ring topology and requires much less material than NMR studies (16). None of these techniques can establish the stereochemistry of the methyllanthionines with absolute confidence. In early studies, this stereochemistry was established for a select few lantibiotics by comparison to chemically synthesized standards in combination with gas chromatography using chiral stationary phases. The stereochemistry determined in these early studies is assumed to be the same for lantibiotics that were discovered and characterized subsequently, but for most members this supposition has not been confirmed experimentally.
Figure 4. Posttranslational modifications in the biosynthesis of the class II lantibiotic lacticin 481. Both the dehydration and the cyclization events are catalyzed by the bifunctional protein LctM. The unmodified leader sequence is removed by the cysteine protease domain of LctT concomitant with transport of the mature lantibiotic outside the cell.
Mode of action
Chemical approaches have also contributed to the current understanding of the mode of action of lantibiotics. Prior to the discovery that nisin and other lantibiotics bind to lipid II, biophysical techniques, such as analysis of efflux of analytes from liposomes and other vesicles, as well as electrophysiology studies had established that many lantibiotics punch holes into membranes (4). More recently, NMR studies have elucidated the details of the molecular recognition used by nisin to sequester lipid II (8), and modification of nisin with fluorescent markers using synthetic chemistry showed that nisin removes lipid II from its functional location in the cell (7). Conversely, fluorescent labeling of lipid II showed that after initial docking of nisin with lipid II a higher order structure is assembled consisting of 4 lipid II molecules and 8 nisin molecules that make up the pores in the bacterial membranes (6).
Several biomimetic studies involving the nonenzymatic, intramolecular cyclization of synthetic peptides that contain dehydroamino acids and unprotected cysteine residues have been used to investigate the cyclization step of lantibiotic biosynthesis. Short peptides that contain a single Dha or Dhb residue and a free cysteine undergo stereoselective cyclization to produce the naturally occurring isomers of (Me)Lan (24-26). These results indicate that the peptides have an innate propensity for Si -face attack on the dehydro amino acids and Re-face protonation of enolates to give the D-configuration at the a-carbons. This selectivity has a kinetic origin and is not caused by thermodynamic control (27). An attempt to prepare biomimetically the nisin A-ring (Lan) and B-ring (MeLan) was unsuccessful because of the much higher reactivity of dehydroalanines compared with dehydrobutyrines (27). Collectively, these studies show that although enzymatic control may not be required for the stereoselectivity of (Me)Lan formation, it is absolutely essential to govern the regioselectivity.
Mechanistic studies on the biosynthetic enzymes
The in vitro reconstitution of the enzymatic activities of LctM, NisC, and EpiD has paved the way to detailed mechanistic studies. The dehydration reaction catalyzed by the bifunctional LctM requires ATP for phosphorylation of the serine and the threonine residues that undergo dehydration (28). Usually, the phosphory- lated intermediate is not observed with the wild type substrate, but incorporation of certain nonproteinogenic amino acids into the substrate peptide by using expressed protein ligation resulted in the accumulation of phosphorylated peptides. Preparation of authentic phosphorylated substrate by ligating synthetic phosphopeptides to a recombinant truncated LctA peptide that carries a C-terminal thioester confirmed that LctM catalyzes the elimination of the phosphate group to generate dehydro amino acids (28). Furthermore, high resolution Fourier transform mass spectrometric analysis of the dehydration reaction has shown that the dehydration reaction takes place by a processive mechanism (29). Mechanistic investigations of the EpiD enzyme that catalyzes the oxidative decarboxylation of the C-terminal cysteine in epidermin biosynthesis to produce the AviCys structure revealed that a (Z)-enethiol is generated that presumably adds to an unsaturated residue in a reaction catalyzed by EpiC (18).
The cloning of the gene clusters involved in the biosynthesis of many lantibiotics laid the foundation for genetic protein engineering aimed at in vivo production of novel compounds with potentially interesting properties. Many studies have indicated the feasibility of changing the molecular structures of lantibiotics by mutagenesis of the prelantibiotic genes (30). In these investigations, not only the biosynthetic machinery, but also the immunity factors had to be considered to generate successful expression systems. Collectively, these studies have demonstrated the low substrate specificity of the biosynthetic enzymes involved. Disadvantages of reprogramming the structures of lantibiotics through in vivo engineering include a limited structural and functional space that can be sampled through mutagenesis and the potential for breakdown of immunity of the producing strain in cases where more active compounds are actually generated. The in vitro reconstitution of the complete biosynthetic pathways for lacticin 481 and haloduracin has provided the opportunity to explore the substrate specificity of their biosynthetic machinery in more detail. Using the expressed protein ligation technology, a series of Ser and Thr analogs embedded in the substrate peptide have been probed as potential substrates for the dehydration reaction, which demonstrates that the dehydratase domain of lacticin 481 synthetase has remarkable substrate promiscuity (31). Similarly, the cyclization domain was shown to tolerate Cys analogs such as selenocysteine, homocysteine, and β3-homocysteine and to convert them to analogs of the naturally occurring (Me)Lan crosslinks (32). In addition to tolerating changes in the structure of the posttranslationally modified amino acids, the enzyme also displays remarkable substrate promiscuity with respect to the position of the dehydrated residues as well as substitutions with nonproteinogenic amino acids at nonmodified positions. Similarly, the oxidative decarboxylase EpiD exhibits a very broad substrate scope (33). These investigations demonstrate that the engineering of novel structures by using the biosynthetic enzymes has great promise.
The Future of Lantibiotics
Many questions still remain with respect to both the biosynthesis and the mode of action of lantibiotics. Whereas the targets of the nisin, mersacidin, and cinnamycin groups are now known, the mechanism of action of many other lantibiotics (e.g., lacticin 481, Pep5, and sublancin) is still unclear. Similarly, the biosynthetic pathways still hold many unresolved questions, which include the molecular recognition that allows the synthetases their high level of substrate promiscuity and at the same time provides exquisite control of the regioselectivity of cyclization. Similarly, the biosynthetic enzymes for the majority of the 15 currently known posttranslational modifications in lantibiotics have not yet been identified, with the rate of discovery of new lantibiotics and new modifications recently outpacing the characterization of new biosynthetic enzymes.
Currently, the most common commercial application for lan- tibiotics is as a preservative in the food industry to combat food-borne pathogens and spoilage bacteria, but many other uses are under active investigation. Duramycin has been shown to increase chloride transport in nasal epithelial cells of cystic fibrosis patients (13), which in turn increases the fluidity of mucus in the lungs and airway and decreases the patient’s susceptibility to infections. Furthermore, several lantibiotics have shown potent activities against multidrug resistant pathogenic bacterial strains. Combined with the development of new techniques to alter the structures of lantibiotics, the future will likely see detailed SAR studies that may result in improved variants.
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