<i>In Vitro</i> Reconstitution and Substrate Specificity of a Lantibiotic Protease

Ihnken, Leigh Anne F.; Chatterjee, Champak; Donk, Wilfred A. van der

doi:10.1021/bi800278n

Cited by 71 publications

(81 citation statements)

References 50 publications

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“…Analysis of the leader peptide of CerA revealed two processing sites, including a GA cleavage site and an additional hexapeptide SDVQPE that were both conserved in ␤ subunits of the two-component lantibiotics cytolysin and lichenicidin (44,45). The putative CerT contained an N-terminal C39 cysteine protease domain, as did LctT and BovT, whose N-terminal 150-aa peptidase domain had been successfully reconstituted in vitro to recognize the double glycine motif (GA/GG) (46,47). Thus, maturation of cerecidins might undergo a two-step process involving complementary function of CerT and CerP.…”

Section: Discussionmentioning

confidence: 99%

Cerecidins, Novel Lantibiotics from Bacillus cereus with Potent Antimicrobial Activity

Wang

Zhang

Teng

et al. 2014

Appl Environ Microbiol

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Lantibiotics are ribosomally synthesized and posttranslationally modified antimicrobial peptides that are widely produced by Gram-positive bacteria, including many species of the Bacillus group. In the present study, one novel gene cluster coding lantibiotic cerecidins was unveiled in Bacillus cereus strain As 1.1846 through genomic mining and PCR screening. The designated cer locus is different from that of conventional class II lantibiotics in that it included seven tandem precursor cerA genes, one modification gene (cerM), two processing genes (cerT and cerP), one orphan regulator gene (cerR), and two immunity genes (cerF and cerE). In addition, one unprecedented quorum sensing component, comQXPA, was inserted between cerM and cerR. The expression of cerecidins was not detected in this strain of B. cereus, which might be due to repressed transcription of cerM. We constitutively coexpressed cerA genes and cerM in Escherichia coli, and purified precerecidins were proteolytically processed with the endoproteinase GluC and a truncated version of putative serine protease CerP. Thus, two natural variants of cerecidins A1 and A7 were obtained which contained two terminal nonoverlapping thioether rings rarely found in lantibiotics. Both cerecidins A1 and A7 were active against a broad spectrum of Gram-positive bacteria. Cerecidin A7, especially its mutant Dhb13A, showed remarkable efficacy against multidrug-resistant Staphylococcus aureus (MDRSA), vancomycin-resistant Enterococcus faecalis (VRE), and even Streptomyces.

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Section: Discussionmentioning

confidence: 99%

Cerecidins, Novel Lantibiotics from Bacillus cereus with Potent Antimicrobial Activity

Wang

Zhang

Teng

et al. 2014

Appl Environ Microbiol

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“…LanT (30,31). However, these studies are challenged by the failure to distinguish between which of the proposed leader peptide functions are affected by the changes in amino acid residues.…”

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confidence: 99%

Biosynthesis and Transport of the Lantibiotic Mutacin 1140 Produced by Streptococcus mutans

et al. 2015

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Lantibiotics are ribosomally synthesized peptide antibiotics composed of an N-terminal leader peptide that is cleaved to yield the active antibacterial peptide. Significant advancements in molecular tools that promote the study of lantibiotic biosynthesis can be used in Streptococcus mutans. Herein, we further our understanding of leader peptide sequence and core peptide structural requirements for the biosynthesis and transport of the lantibiotic mutacin 1140. Our study on mutacin 1140 biosynthesis shows a dedicated secondary cleavage site within the leader peptide and the dependency of transport on core peptide posttranslational modifications (PTMs). The secondary cleavage site on the leader peptide is found at the ؊9 position, and secondary cleavage occurs before the core peptide is transported out of the cell. The coordinated cleavage at the ؊9 position was absent in a lanT deletion strain, suggesting that the core peptide interaction with the LanT transporter enables uniform cleavage at the ؊9 position. Following transport, the LanP protease was found to be tolerant to a wide variety of amino acid substitutions at the primary leader peptide cleavage site, with the exception of arginine at the ؊1 position. Several leader and core peptide mutations produced core peptide variants that had intermediate stages of PTM enzyme modifications, supporting the concept that PTM enzyme modifications, secondary cleavage, and transport are occurring in a highly coordinated fashion. IMPORTANCEMutacin 1140 belongs to the class I lantibiotic family of ribosomally synthesized and posttranslationally modified peptides (RiPPs). The biosynthesis of mutacin 1140 is a highly efficient process which does not lead to a discernible level of production of partially modified core peptide variants. The products isolated from an extensive mutagenesis study on the leader and core peptides of mutacin 1140 show that the posttranslational modifications (PTMs) on the core peptide occur under a highly coordinated dynamic process. PTMs are dictated by the distance of the core peptide modifiable residues from PTM enzyme active sites. The formation of lanthionine rings aids in the formation of successive PTMs, as was observed in a peptide variant lacking a Cterminal decarboxylation. Lantibiotics are a class of ribosomally synthesized peptide antibiotics produced by Gram-positive bacteria, such as Lactococcus lactis and Streptococcus mutans (1, 2). Lantibiotics are characterized by the presence of posttranslational modifications (PTMs), such as dehydrated residues and lanthionine rings. The modified residues 2,3-didehydroalanine (Dha) and 2,3-didehydrobutyrine (Dhb) are formed from dehydration of serines and threonines, respectively. The cyclization between a cysteine and either a Dha or a Dhb forms a lanthionine or a methyllanthionine ring, respectively. The biosynthetic gene cluster contains all the genes necessary to produce a lantibiotic. The lan operon for class I lantibiotics contains genes encoding the lantibiotic peptide (lanA); the...

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“…The purified PEP of LctT can cleave off the leader peptide of both the modified and unmodified precursor peptide (9). On the other hand, we previously reported that full-length NukT can only digest modified NukA (15).…”

Section: Discussionmentioning

confidence: 97%

“…Members of this ABC transporter subfamily are involved in the secretion of proteinaceous compounds and contain an additional N-terminal domain involved in the processing of their substrates at the so-called double glycine site at the C terminus of the leader peptide (7)(8)(9). There are only a few studies on the N-terminal peptidase domain (PEP) of AMS proteins.…”

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confidence: 99%

Lantibiotic Transporter Requires Cooperative Functioning of the Peptidase Domain and the ATP Binding Domain

Nishie

Sasaki

Nagao

et al. 2011

Journal of Biological Chemistry

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Lantibiotics are lanthionine-containing antibiotic peptides, which display a number of favorable characteristics. For example, they have no taste or odor, they are tolerant to high heat and acid, and they have stable activity at nanomolar concentrations against antibiotic-resistant pathogens. These attractive qualities are expected to lead to the broad application potentials of lantibiotics (1-4). One of the best known lantibiotics, nisin, which is produced by Lactococcus lactis, has been used as a food preservative for decades. Lantibiotics are divided into two different classes based on the biosynthetic pathway and the enzymes from which they are produced (5, 6). Ribosomally synthesized prepeptide (LanA; "Lan" is an abbreviation for enzymes and/or proteins related to lantibiotic biosynthesis) is inactive and consists of an N-terminal leader peptide and a C-terminal propeptide portion. In class I lantibiotics such as nisin, LanB dehydrates serine and threonine residues of the propeptide, LanC cyclizes dehydrated residues with cysteine, LanT secretes the peptide, and the extracellular protein LanP cleaves off the N-terminal leader peptide. In class II lantibiotics such as nukacin ISK-1, a single enzyme, LanM, catalyzes both dehydration and cyclization, and ABC 2 transporter LanT removes the leader peptide concomitantly with secretion.LanT of the class II lantibiotics is a member of the ABC transporter maturation and secretion (AMS) protein family. Members of this ABC transporter subfamily are involved in the secretion of proteinaceous compounds and contain an additional N-terminal domain involved in the processing of their substrates at the so-called double glycine site at the C terminus of the leader peptide (7-9). There are only a few studies on the N-terminal peptidase domain (PEP) of AMS proteins. The PEPs of LagD, CvaB, ComA, and LctT have been purified and been proven to cleave off the leader peptide of their cognate precursor peptide (7, 9 -11). The PEPs of these proteins have Cys, His, or Asp residues, which are conserved among the papain-like cysteine proteases. Substitution of these residues results in loss of their peptidase activity, demonstrating that PEP also belongs to the cysteine protease family (9 -11). The N-terminal leader peptide sequence of the precursor peptide has been reported to be important for peptidase activity of PEP. Furgerson Ihnken et al. found that the double glycine motif and the ␣-helical structure of the leader peptide are important for proteolysis by PEP of LctT (9). Kotake et al. also demonstrated that PEP of ComA cleaves off the leader peptide after the double glycine site and proposed the presence of hydrophobic patch on the surface of the PEP, which interacts with the hydrophobic face of the helix of the precursor peptide (12). A three-dimensional crystal structure of ComA has been recently clarified, and PEP was

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In Vitro Reconstitution and Substrate Specificity of a Lantibiotic Protease

Cited by 71 publications

References 50 publications

Cerecidins, Novel Lantibiotics from Bacillus cereus with Potent Antimicrobial Activity

Cerecidins, Novel Lantibiotics from Bacillus cereus with Potent Antimicrobial Activity

Biosynthesis and Transport of the Lantibiotic Mutacin 1140 Produced by Streptococcus mutans

Lantibiotic Transporter Requires Cooperative Functioning of the Peptidase Domain and the ATP Binding Domain

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