Structure of the nisin leader peptidase NisP revealing a C-terminal autocleavage activity

Xu, Yueyang; Li, Xin; Li, Ruiqing; Li, Shanshan; Ni, Hongqian; Wang, Hui; Xu, Haijin; Zhou, Weihong; Saris, Per E. J.; Yang, Wen; Qiao, Mingqiang; Rao, Zihe

doi:10.1107/s1399004714004234

Cited by 28 publications

(39 citation statements)

References 38 publications

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“…Furthermore, the recognition of a region upstream of S(Ϫ8) is not likely, given that the leader peptide is only 8 amino acids and given the presence of the secondary cleavage site. Core peptide recognition by NisP for the cleavage of the leader peptide has been suggested for nisin (7,22,45), and given the current results, core peptide structure is important for the specificity of mutacin 1140 leader peptide cleavage by LanP. Additional studies will need to be done to determine whether LanP can recognize unmodified core peptide or whether PTM enzyme modifications are important for the recognition.…”

Section: Discussionmentioning

confidence: 82%

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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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Section: Discussionmentioning

confidence: 82%

“…The substrate specificity of LanP varies among lantibiotics (6). In most lantibiotic systems, LanP is believed to be associated with the extracellular leaflet of the membrane and cell wall (7). Therefore, cleavage does not occur until mutacin 1140 is transported out of the cell by LanT.…”

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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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“…334 The C-terminal anchoring peptide can also be excised from NisP by autoproteolysis, likely freeing it from the cell wall. 335 …”

Section: Proteases and Export In Class I And Ii Lanthipeptidesmentioning

confidence: 99%

“…335 Although the sequence of the prodomain was included in the protein construct used for crystallization, the reported structure consists only of the serine protease catalytic domain composed of Ser224 through Arg566. SDS-PAGE analysis of dissolved crystals revealed that the protein had undergone proteolytic degradation in situ, facilitating crystallization of the resultant fragment consisting of residues 224–566.…”

Section: Proteases and Export In Class I And Ii Lanthipeptidesmentioning

confidence: 99%

Mechanistic Understanding of Lanthipeptide Biosynthetic Enzymes

et al. 2017

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Lanthipeptides are ribosomally synthesized and post-translationally modified peptides (RiPPs) that display a wide variety of biological activities, from antimicrobial to antiallodynic. Lanthipeptides that display antimicrobial activity are called lantibiotics. The post-translational modification reactions of lanthipeptides include dehydration of Ser and Thr residues to dehydroalanine and dehydrobutyrine, a transformation that is carried out in three unique ways in different classes of lanthipeptides. In a cyclization process, Cys residues then attack the dehydrated residues to generate the lanthionine and methyllanthionine thioether cross-linked amino acids from which lanthipeptides derive their name. The resulting polycyclic peptides have constrained conformations that confer their biological activities. After installation of the characteristic thioether cross-links, tailoring enzymes introduce additional post-translational modifications that are unique to each lanthipeptide and that fine-tune their activities and/or stability. This review focuses on studies published over the past decade that have provided much insight into the mechanisms of the enzymes that carry out the post-translational modifications.

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“…In the maturation of subtilin, no specific protease has been found and the processing takes place outside the cell probably by diverse serine proteases (Corvey et al, 2003 ). The first lantibiotic protease with a resolved 3D structure, EpiP from Staphylococcus aureus , an analog of NisP, has been reported (Kuhn et al, 2014 ; Xu et al, 2014 ). On the other hand, in type II lanthipeptides, the protease domain is fused to the transporter and this protein cleaves behind a double glycine motif (Knerr and van der Donk, 2012 ).…”

Section: Introductionmentioning

confidence: 99%

Specificity and Application of the Lantibiotic Protease NisP

et al. 2018

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Lantibiotics are ribosomally produced and posttranslationally modified peptides containing several lanthionine residues. They exhibit substantial antimicrobial activity against Gram-positive bacteria, including relevant pathogens. The production of the model lantibiotic nisin minimally requires the expression of the modification and export machinery. The last step during nisin maturation is the cleavage of the leader peptide. This liberates the active compound and is catalyzed by the cell wall-anchored protease NisP. Here, we report the production and purification of a soluble variant of NisP. This has enabled us to study its specificity and test its suitability for biotechnological applications. The ability of soluble NisP to cleave leaders from various substrates was tested with two sets of nisin variants. The first set was designed to investigate the influence of amino acid variations in the leader peptide or variations around the cleavage site. The second set was designed to study the influence of the lanthionine ring topology on the proteolytic efficiency. We show that the substrate promiscuity is higher than has previously been suggested. Our results demonstrate the importance of the arginine residue at the end of the leader peptide and the importance of lanthionine rings in the substrate for specific cleavage. Collectively, these data indicate that NisP is a suitable protease for the activation of diverse heterologously expressed lantibiotics, which is required to release active antimicrobial compounds.

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Structure of the nisin leader peptidase NisP revealing a C-terminal autocleavage activity

Cited by 28 publications

References 38 publications

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

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

Mechanistic Understanding of Lanthipeptide Biosynthetic Enzymes

Specificity and Application of the Lantibiotic Protease NisP

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