Structure-function relationships of the lanthionine cyclase SpaC involved in biosynthesis of the Bacillus subtilis peptide antibiotic subtilin

Biosynthesis of the lantibiotic subtilin in Bacillus subtilis is accomplished by a synthetase complex consisting of the dehydratase SpaB, cyclase SpaC, and transporter SpaT. Genetically engineered subtilin cyclases SpaC and related NisC and EriC proteins involved in biosynthesis of the lantibiotics nisin and ericin A/S, respectively, were analyzed to functionally substitute native SpaC in vivo. We could show for the first time posttranslational modification of a lantibiotic precursor peptide (subtilin) by a hybrid lantibiotic synthetase (SpaBT/EriC). Genetically engineered SpaC alanine replacement mutants revealed the essentiality of residues His231, Trp302, Cys303, Tyr304, Gly305, Cys349, and His350, as well as the conserved C-terminal motif Lys437-Ala438-Leu439-Leu440-Ile441 for subtilin biosynthesis. Assignment of these strictly conserved lantibiotic cyclase residues to the NisC structure [Li, B., Yu, J. B., Brunzelle, J. S., Moll, G. N., van der Donk, W. A., and Nair, S. K. (2006) Science, 311, 1464-1467] revealed the first experimental evidence for structure-function relationships in catalytic centers of lantibiotic cyclases. SpaC residues His231, Cys303, and Cys349 are involved in coordination of the central zinc ion. The pair His231/Tyr304 is discussed to act as general acid/base catalysts in lanthionine formation. Furthermore, pull-down experiments revealed that functional inactive SpaC mutants were still able to interact with the hexahistidine-tagged subtilin precursor peptide in vitro. Our results suggest that Trp302 and the C-terminal residues of SpaC are constituents of a hydrophobic cluster which is involved in stabilization of the catalytic center and binding of the subtilin precursor peptide.     

The biosynthesis of the lantibiotics epidermin,gallidermin, Pep5 and epilancin K7

Lantibiotics are antibiotic peptides that contain the rare thioether amino acids lanthionine and/or methyllanthionine. Epidermin, Pep5 and epilancin K7 are produced by Staphylococcus epidermidis whereas gallidermin (6L-epidermin) was isolated from the closely related species Staphylococcus gallinarum. The biosynthesis of all four lantibiotics proceeds from structural genes which code for prepeptides that are enzymatically modified to give the mature peptides. The genes involved in biosynthesis, processing, export etc. are found in gene clusters adjacent to the structural genes and code for transporters, immunity functions, regulatory proteins and the modification enzymes LanB, LanC and LanD, which catalyze the biosynthesis of the rare amino acids. LanB and LanC are responsible for the dehydration of the serine and threonine residues to give dehydroalanine and dehydrobutyrine and subsequent addition of cysteine SH-groups to the dehydro amino acids which results in the thioether rings. EpiD, the only LanD enzyme known so far, catalyzes the oxidative decarboxylation of the C-terminal cysteine of epidermin which gives the C-terminal S-aminovinylcysteine after addition of a dehydroalanine residue.Abbreviations Dha 2,3-didehydroalanine - Dhb 2,3-didehydrobutyrine - Lan lanthionine - Melan methyllanthionine     

Microbial engineering of dehydro-amino acids and lanthionines in non-lantibiotic peptides

This minireview focusses on the use of bacteria to introduce dehydroresidues and (methyl)lanthionines in (poly)peptides. It mainly describes the broad exploitation of bacteria containing lantibiotic enzymes for the engineering of these residues in a wide variety of peptides in particular in peptides unrelated to lantibiotics. Lantibiotic dehydratases dehydrate serines and threonines present in peptides preceded by a lantibiotic leader peptide thus forming dehydroalanine and dehydrobutyrine, respectively. These dehydroresidues can be coupled to cysteines thus forming (methyl)lanthionines. This coupling is catalysed by lantibiotic cyclases. The design, synthesis, and export of microbially engineered dehydroresidue and or lanthionine-containing peptides in non-lantibiotic peptides are reviewed, illustrated by some examples which demonstrate the high relevance of these special residues. This minireview is the first with special focus on the microbial engineering of nonlantibiotic peptides by exploiting lantibiotic enzymes.     

Genetics of subtilin and nisin biosyntheses

Several peptide antibiotics have been described as potent inhibitors of bacterial growth. With respect to their biosynthesis, they can be devided into two classes: (i) those that are synthesized by a non-ribosomal mechanism and (ii) those that are ribosomally synthesized. Subtilin and nisin belong to the ribosomally synthesized peptide antibiotics. They contain the rare amino acids dehydroalanine, dehydrobutyrine, meso-lanthionine, and 3-methyl-lanthionine. They are derived from prepeptides which are post-translationally modiffied and have been termed lantibiotics because of their characteristic lanthionine bridges (Schnell et al. 1988). Nisin is the most prominent lantibiotic and is used as a food preservative due to its high potency against certain gram-positive bacteria (Mattick & Hirsch 1944, 1947; Rayman & Hurst 1984). It is produced by Lactococcus lactis strains belonging to serological group N. The potent bactericidal activities of nisin and other lantibiotics are based on depolarization of energized bacterial cytoplasmic membranes. Breakdown of the membrane potential is initiated by the formation of pores through which molecules of low molecular weight are released. A trans-negative membrane potential of 50 to 100 mV is necessary for pore formation by nisin (Ruhr & Sahl 1985; Sahl et al. 1987). Nisin occurs as a partially amphiphilic molecule (Van de Ven et al. 1991). Apart from the detergent-like effect of nisin on cytoplasmic membranes, an inhibition of murein synthesis has also been discussed as the primary effect (Reisinger et al. 1980). In several countries nisin is used to prevent the growth of clostridia in cheese and canned food. The nisin peptide structure was first described by Gross & Morall (1971), and its structural gene was isolated in 1988 (Buchman et al. 1988; Kaletta & Entian 1989). Nisin has two natural variants, nisin A and nisin Z, which differ in a single amino acid residue at position 27 (histidin in nisin A is replaced by asparagin in nisin Z (Mulders et al. 1991; De Vos et al. 1993). Subtilin is produced by Bacillus subtilis ATCC 6633. Its chemical structure was first unravelled by Gross & Kiltz (1973) and its structural gene was isolated in 1988 (Banerjee & Hansen 1988). Subtilin shares strong similarities to nisin with an identical organization of the lanthionine ring structures (Fig. 1), and both lantibiotics possess similar antibiotic activities. Due to its easy genetic analysis B. subtilis became a very suitable model organism for the identification and characterization of genes and proteins involved in lantibiotic biosynthesis. The pathway by which nisin is produced is very similar to that of subtilin, and the proteins involved share significant homologies over the entire proteins (for review see also De Vos et al. 1995b). The respective genes have been identified adjacent to the structural genes, and are organized in operon-like structures (Fig. 2). These genes are responsible for post-translational modification, transport of the modified prepeptide, proteolytic cleavage, and immunity which prevents toxic effects on the producing bacterium. In addition to this, biosynthesis of subtilin and nisin is strongly regulated by a two-component regulatory system which consists of a histidin kinase and a response regulator protein.     

Engineering dehydrated amino acid residues in the antimicrobial peptide nisin.

The small antimicrobial peptide nisin, produced by Lactococcus lactis, contains the uncommon amino acid residues dehydroalanine and dehydrobutyrine and five thio ether bridges. Since these structures are posttranslationally formed from Ser, Thr, and Cys residues, it is feasible to study their role in nisin function and biosynthesis by protein engineering. Here we report the development of an expression system for mutated nisin Z (nisZ) genes, using nisin A producing L. lactis as a host. Replacement by site-directed mutagenesis of the Ser-5 codon in nisZ by a Thr codon, led to a mutant with a dehydrobutyrine instead of a dehydroalanine residue at position 5, as shown by NMR. Its antimicrobial activity was 2-10-fold lower relative to wild-type nisin Z, depending on the indicator strain used. In another mutagenesis study a double mutation was introduced in the nisZ gene by replacing the codons for Met-17 and Gly-18 by codons for Gln and Thr, respectively, as in the third lanthionine ring of the related antimicrobial peptide subtilin from Bacillus subtilis. This resulted in the simultaneous production of two mutant species, one containing a Thr residue and the other containing a dehydrobutyrine residue at position 18, both having different bacteriocidal properties.     

The number and nature of , -unsaturated amino acids in subtilin

In subtilin, a peptide produced by Bacillus subtilis, there are present three α,β-unsaturated amino acids, namely, two residues of dehydroalanine and one residue of β-methyldehydroalanine (dehydrobutyrine). Subtilin and nisin, a polypeptide produced by Streptococcus lactis, thus have in common not only the COOH-terminal sequence dehydroalanyllysine but also the number and nature of α,β-unsaturated amino acids.     

Lantibiotic Reductase LtnJ Substrate Selectivity Assessed with a Collection of Nisin Derivatives as Substrates

Lantibiotics are potent antimicrobial peptides characterized by the presence of dehydrated amino acids, dehydroalanine and dehydrobutyrine, and (methyl)lanthionine rings. In addition to these posttranslational modifications, some lantibiotics exhibit additional modifications that usually confer increased biological activity or stability on the peptide. LtnJ is a reductase responsible for the introduction of d-alanine in the lantibiotic lacticin 3147. The conversion of l-serine into d-alanine requires dehydroalanine as the substrate, which is produced in vivo by the dehydration of serine by a lantibiotic dehydratase, i.e., LanB or LanM. In this work, we probe the substrate specificity of LtnJ using a system that combines the nisin modification machinery (dehydratase, cyclase, and transporter) and the stereospecific reductase LtnJ in Lactococcus lactis. We also describe an improvement in the production yield of this system by inserting a putative attenuator from the nisin biosynthesis gene cluster in front of the ltnJ gene. In order to clarify the sequence selectivity of LtnJ, peptides composed of truncated nisin and different mutated C-terminal tails were designed and coexpressed with LtnJ and the nisin biosynthetic machinery. In these tails, serine was flanked by diverse amino acids to determine the influence of the surrounding residues in the reaction. LtnJ successfully hydrogenated peptides when hydrophobic residues (Leu, Ile, Phe, and Ala) were flanking the intermediate dehydroalanine, while those in which dehydroalanine was flanked by one or two polar residues (Ser, Thr, Glu, Lys, and Asn) or Gly were either less prone to be modified by LtnJ or not modified at all. Moreover, our results showed that dehydrobutyrine cannot serve as a substrate for LtnJ.     

Functional and structural aspects of poplar cytosolic and plastidial type a methionine sulfoxide reductases

The genome of Populus trichocarpa contains five methionine sulfoxide reductase A genes. Here, both cytosolic (cMsrA) and plastidial (pMsrA) poplar MsrAs were analyzed. The two recombinant enzymes are active in the reduction of methionine sulfoxide with either dithiothreitol or poplar thioredoxin as a reductant. In both enzymes, five cysteines, at positions 46, 81, 100, 196, and 202, are conserved. Biochemical and enzymatic analyses of the cysteine-mutated MsrAs support a catalytic mechanism involving three cysteines at positions 46, 196, and 202. Cys(46) is the catalytic cysteine, and the two C-terminal cysteines, Cys(196) and Cys(202), are implicated in the thioredoxin-dependent recycling mechanism. Inspection of the pMsrA x-ray three-dimensional structure, which has been determined in this study, strongly suggests that contrary to bacterial and Bos taurus MsrAs, which also contain three essential Cys, the last C-terminal Cys(202), but not Cys(196), is the first recycling cysteine that forms a disulfide bond with the catalytic Cys(46). Then Cys(202) forms a disulfide bond with the second recycling cysteine Cys(196) that is preferentially reduced by thioredoxin. In agreement with this assumption, Cys(202) is located closer to Cys(46) compared with Cys(196) and is included in a (202)CYG(204) signature specific for most plant MsrAs. The tyrosine residue corresponds to the one described to be involved in substrate binding in bacterial and B. taurus MsrAs. In these MsrAs, the tyrosine residue belongs to a similar signature as found in plant MsrAs but with the first C-terminal cysteine instead of the last C-terminal cysteine.     

NisC, the cyclase of the lantibiotic nisin, can catalyze cyclization of designed nonlantibiotic peptides

Nisin is a pentacyclic peptide antibiotic active against Gram-positive bacteria. Its thioether rings are formed by two enzymatic steps: nisin dehydratase (NisB)-mediated dehydration of serines and threonines followed by nisin cyclase (NisC)-catalyzed enantioselective coupling of cysteines to the formed dehydroresidues. Here, we report the in vivo activity of NisC to cyclize a wide array of unrelated and designed peptides that were fused to the nisin leader peptide. To assess the role of NisC, leader peptide fusions, secreted by Lactococcus lactis cells containing NisBT with or without NisC were compared. In hexapeptides, a dehydroalanine could spontaneously react with a more C-terminally located cysteine. In contrast, peptides containing dehydrobutyrines require NisC for cyclization. In agreement with in silico predictions NisC could efficiently cyclize the hexapeptides ADhbVECK and IDhbPGCK, but ADhbVWCE was not cyclized. Interestingly, NisC could efficiently catalyze the synthesis of peptides with intertwined rings and of a designed polyhexapeptide containing four thioether rings. Taken together the data demonstrate that NisC can be widely applied for the cyclization and stabilization of nonlantibiotic peptides.     

New developments in lantibiotic biosynthesis and mode of action

Lantibiotics are a unique class of peptide antibiotics. Recent studies of the proteins involved in the elaborate post-translational modifications of lantibiotics have revealed that these enzymes have relaxed substrate specificity. These modifications include the dehydration of serine and threonine residues followed by the intramolecular addition of cysteine thiols to the unsaturated amino acids to create an intricate polycyclic peptide. The use of peptide engineering in vivo and in vitro has allowed investigation of their biosynthetic machinery. Several members utilize a unique mode of biological action that involves the sequestration of lipid II, a crucial intermediate in peptidoglycan biosynthesis, to form pores in bacterial membranes.     

Mechanistic investigations of the dehydration reaction of lacticin 481 synthetase using site-directed mutagenesis

Lantibiotic synthetases catalyze the dehydration of Ser and Thr residues in their peptide substrates to dehydroalanine (Dha) and dehydrobutyrine (Dhb), respectively, followed by the conjugate addition of Cys residues to the Dha and Dhb residues to generate the thioether cross-links lanthionine and methyllanthionine, respectively. In this study ten conserved residues were mutated in the dehydratase domain of the best characterized family member, lacticin 481 synthetase (LctM). Mutation of His244 and Tyr408 did not affect dehydration activity with the LctA substrate whereas mutation of Asn247, Glu261, and Glu446 considerably slowed down dehydration and resulted in incomplete conversion. Mutation of Lys159 slowed down both steps of the net dehydration: phosphorylation of Ser/Thr residues and the subsequent phosphate elimination step to form the dehydro amino acids. Mutation of Arg399 to Met or Leu resulted in mutants that had phosphorylation activity but displayed greatly decreased phosphate elimination activity. The Arg399Lys mutant retained both activities, however. Similarly, the Thr405Ala mutant phosphorylated the LctA substrate but had compromised elimination activity. Finally, mutation of Asp242 or Asp259 to Asn led to mutant enzymes that lacked detectable dehydration activity. Whereas the Asp242Asn mutant retained phosphate elimination activity, the Asp259Asn mutant was not able to eliminate phosphate from a phosphorylated substrate peptide. A model is presented that accounts for the observed phenotypes of these mutant enzymes.     

Zinc environment in sheep liver sorbitol dehydrogenase

The extended X-ray absorption fine structure (EXAFS) associated with the zinc K-absorption edge has been recorded for sorbitol dehydrogenase. It is interpreted in terms of one cysteine sulfur among the ligands to the active site zinc atom. Simulations of the EXAFS based on the presence of two such sulfurs are less satisfactory, and comparison with the EXAFS of such systems points to the presence of only one sulfur ligand in sorbitol dehydrogenase. These results provide evidence that sorbitol dehydrogenase does not have the characteristic one water, one His, two Cys arrangement of ligands to the active site zinc found in the homologous alcohol dehydrogenases and are consistent with the one water, one His, one Cys, one Glu ligand arrangement of the proposed model of sorbitol dehydrogenase [Eklund, H., Horjales, E., J?rnvall, H., Br?ndén, C.-I., & Jeffery J. (1985) Biochemistry 24, 8005-8012]. Evidence for the correctness of the model is also evidence for validity of predictive techniques used in constructing the model, i.e., computer graphics fitting of the amino acid sequence to the crystallographically derived structure of a different but homologous protein.     

Structures of lantibiotics studied by NMR

During the last decade, Nuclear Magnetic Resonance has played an important role in the unravelling of the primary and tertiary structures of lantibiotics. A short overview of these studies, together with typical spatial structures obtained, is presented.Abbreviations Ala _S D-alanyl moiety of meso-lanthionine - _S Ala L-alanyl moiety of meso-lanthionine - Abu 3-methylalanyl moiety of (2S3S,6R)-3-methyllanthionine - Dha dehydroalanine - Dhb dehydrobutyrine - DPC docecylphosphocholine - NMR Nuclear Magnetic Resonance - NOE Nuclear Overhauser Enhancement - NOESY NOE spectroscopy - TOCSY Total Correlation Spectroscopy     

Introduction of selective intersubunit disulfide bonds into self‐assembly protein scaffold to enhance an artificial multienzyme complex's activity

In nature, many enzymes participating in multienzyme reactions are often assembled to enhance efficiencies of multiple reactions. Therefore, much attention has been focused on self‐assembly of multiple enzymes fused with a protein/peptide that interacts with a specific protein to enhance artificial multienzyme reactions. Sulfolobus solfataricus proliferating cell nuclear antigen (PCNA) is a ring‐shaped symmetric heterotrimer consisting of PCNA1, PCNA2 and PCNA3. Multiple enzymes can be co‐localized on the PCNA ring by fusing them to the C‐termini of the three PCNA subunits. However, an advantage of the specific non‐covalent complex formation is inextricably associated with the disadvantage of its concentration‐dependent dissociation. In this study, disulfide bonds were introduced between the PCNA subunits by Cys substitution at the sites neighboring the interface for heterotrimerization. Selective intersubunit disulfide bond formation between PCNA1 and PCNA3 and between PCNA2 and PCNA3 by a natural oxidizing reagent successfully stabilized an artificial multienzyme complex, which is composed of a bacterial cytochrome P450 and its two redox partner proteins. The covalent stabilization of the multienzyme complex enhanced its cytochrome P450 activity because of the absence of inactive dissociated components. Biotechnol. Bioeng. 2013; 110: 1858–1864. © 2013 Wiley Periodicals, Inc.     

乳链菌肽Nisin的生物合成及表达调控机制

乳链菌肽Nisin是乳酸乳球菌（Lactococcus lactis）产生的一种多肽类抗菌物质,是一种由34个氨基酸组成的羊毛硫细菌素。与Nisin合成有关的基因有11个,构成一个基因簇nisA（Z）BTCIPRKFEG。这些相关基因组成三个操纵子进行转录,分别是nisA（Z）BTCIP、nisRK和nisFEG。Nisin通过NisRK双组分调节系统诱导自身合成,而NisI和NisFEG赋予了Nisin产生菌对Nisin的免疫性。对于Nisin的生物合成机制人们展开了非常广泛的研究。本文对Nisin的结构、Nisin合成相关的基因簇、Nisin的生物合成及表达调控机制以及Nisin产生菌对Nisin的免疫性进行了综述。     

Some chemical and physical properties of nisin, a small-protein antibiotic produced by Lactococcus lactis

Nisin is a small gene-encoded antimicrobial protein produced by Lactococcus lactis that contains unusual dehydroalanine and dehydrobutyrine residues. The reactivity of these residues toward nucleophiles was explored by reacting nisin with a variety of mercaptans. The kinetics of reaction with 2-mercaptoethane-sulfonate and thioglycolate indicated that the reaction pathway includes a binding step. Reaction of nisin at high pH resulted in the formation of multimeric products, apparently as a result of intramolecular and intermolecular reactions between nucleophilic groups and the dehydro residues. One of the nucleophiles had a pKa of about 9.8. The unique vinyl protons of the dehydro residues that give readily identifiable proton nuclear magnetic resonances were used to observe the addition of nucleophiles to the dehydro moiety. After reaction with nucleophiles, nisin lost its antibiotic activity and no longer showed the dehydro resonances, indicating that the dehydro groups had been modified. The effect of pH on the solubility of nisin was determined; the solubility was quite high at low pH (57 mg/ml at pH 2) and was much lower at high pH (0.25 mg/ml at pH 8 to 12), as measured before significant pH-induced chemical modification had occurred. High-performance liquid chromatography on a C18 column was an effective technique for separating unmodified nisin from its reaction products. The cyanogen bromide cleavage products of nisin were about 90% less active toward inhibition of bacterial spore outgrowth than was native nisin. These results are consistent with earlier observations, which suggested that the dehydro residues of nisin have a role in the mechanism of antibiotic action, in which they act as electrophilic Michael acceptors toward nucleophiles in the cellular target.     

Some chemical and physical properties of nisin, a small-protein antibiotic produced by Lactococcus lactis.

Nisin is a small gene-encoded antimicrobial protein produced by Lactococcus lactis that contains unusual dehydroalanine and dehydrobutyrine residues. The reactivity of these residues toward nucleophiles was explored by reacting nisin with a variety of mercaptans. The kinetics of reaction with 2-mercaptoethane-sulfonate and thioglycolate indicated that the reaction pathway includes a binding step. Reaction of nisin at high pH resulted in the formation of multimeric products, apparently as a result of intramolecular and intermolecular reactions between nucleophilic groups and the dehydro residues. One of the nucleophiles had a pKa of about 9.8. The unique vinyl protons of the dehydro residues that give readily identifiable proton nuclear magnetic resonances were used to observe the addition of nucleophiles to the dehydro moiety. After reaction with nucleophiles, nisin lost its antibiotic activity and no longer showed the dehydro resonances, indicating that the dehydro groups had been modified. The effect of pH on the solubility of nisin was determined; the solubility was quite high at low pH (57 mg/ml at pH 2) and was much lower at high pH (0.25 mg/ml at pH 8 to 12), as measured before significant pH-induced chemical modification had occurred. High-performance liquid chromatography on a C18 column was an effective technique for separating unmodified nisin from its reaction products. The cyanogen bromide cleavage products of nisin were about 90% less active toward inhibition of bacterial spore outgrowth than was native nisin. These results are consistent with earlier observations, which suggested that the dehydro residues of nisin have a role in the mechanism of antibiotic action, in which they act as electrophilic Michael acceptors toward nucleophiles in the cellular target.     

Extended X-ray absorption fine structure of copper in CuA-depleted, p-(hydroxymercuri)benzoate-modified, and native cytochrome c oxidase

Cytochrome c oxidase contains four redox-active metal centers: two heme irons, cytochromes a and a3, and two copper ions, CuA and CuB. Due to the paucity of spectroscopic signatures for both copper sites in cytochrome c oxidase, the ligands and structures for these sites have remained ambiguous. The specific depletion of CuA from the p-(hydroxymercuri)benzoate- (pHMB-) modified cytochrome c oxidase recently reported [Gelles, J., & Chan, S. I. (1985) Biochemistry 24, 3963-3972] is herein described. Characterization of this enzyme shows that the structures of the remaining metal centers are essentially unperturbed by the CuA modification and depletion (P. M. Li, J. Gelles, and S. I. Chan, unpublished results). Copper extended X-ray absorption fine structure (EXAFS) measurements on the CuA-depleted cytochrome c oxidase reveal coordination of three (N, O) ligands and one (S, Cl) ligand at the CuB site. Comparison of EXAFS results obtained for the CuA-depleted, pHMB-modified, and "unmodified control" enzymes has allowed the deconvolution of the EXAFS in terms of the inner coordination spheres for CuA as well as CuB. On the basis of these data, it is found that the structure for the CuA site is consistent with two (N, O) ligands and two S ligands.