Effect of amino acid substitutions in conserved residues in the leader peptide on biosynthesis of the lantibiotic mutacin II

Chen, Ping; Qi, Feng Xia; Novák, Jan; Krull, Robert; Caufield, Page W.

doi:10.1111/j.1574-6968.2001.tb10511.x

Cited by 38 publications

(40 citation statements)

References 18 publications

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“…3), including streptococcin A-FF22 (SA-FF22), lacticin 481, variacin (Pridmore et al, 1996), mutacin II (Woodruff et al, 1998) and salivaricin A (Ross et al, 1993). It contains a GG (double-glycine) motif immediately preceding the cleavage site, and a conserved EL sequence (Sablon et al, 2000;Chen et al, 2001). The leader peptide of bovicin HJ50 contains a TVS motif instead of the conserved EVT/EVS sequences.…”

Section: Analysis Of the Gene Encoding Bovicin Hj50mentioning

confidence: 99%

Bovicin HJ50, a novel lantibiotic produced by Streptococcus bovis HJ50

Xiao

Chen

et al. 2004

Microbiology

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A bacteriocin-producing strain was isolated from raw milk and named Streptococcus bovis HJ50. Like most bacteriocins produced by lactic acid bacteria, bovicin HJ50 showed a narrow range of inhibiting activity. It was sensitive to trypsin, subtilisin and proteinase K. Bovicin HJ50 was extracted by n-propanol and purified by SP Sepharose Fast Flow, followed by Phenyl Superose and Sephadex G-50. Treatment of Micrococcus flavus NCIB8166 with bovicin HJ50 revealed potassium efflux from inside the cell in a concentration-dependent manner. The molecular mass of bovicin HJ50 was determined to be 3428?3 Da. MS analysis of DTT-treated bovicin HJ50 suggested that bovicin HJ50 contains a disulfide bridge. The structural gene of bovicin HJ50 was cloned by nested PCR based on its N-terminal amino acid sequence. Sequence analysis showed that it encodes a 58 aa prepeptide consisting of an N-terminal leader sequence of 25 aa and a C-terminal propeptide domain of 33 aa. Bovicin HJ50 shows similarity to type AII lantibiotics. Chemical modification using an ethanethiol-containing reaction mixture showed that two Thr residues are modified.

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Section: Analysis Of the Gene Encoding Bovicin Hj50mentioning

confidence: 99%

Bovicin HJ50, a novel lantibiotic produced by Streptococcus bovis HJ50

Xiao

Chen

et al. 2004

Microbiology

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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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“…It may act in a protective role for the producing organism, as its removal is required for bioactivity (10)(11)(12)(13)(14)(15). The leader peptide could also serve as a recognition motif for the transport machinery because non-lantibiotic peptides attached to leader peptides are secreted (16,17), or as a scaffold for the lantibiotic synthetases (11,(18)(19)(20).…”

mentioning

confidence: 99%

“…With the exception of a very recent report on ComC (27), these previous studies focused mainly on characterization of the protease with investigation of substrate recognition receiving little attention. The study of lantibiotic proteases has been restricted to limited in vivo mutagenesis of the leader peptides (10,13,15,28,29).…”

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confidence: 99%

In Vitro Reconstitution and Substrate Specificity of a Lantibiotic Protease

2008

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Lacticin 481 is a lanthionine-containing bacteriocin (lantibiotic) produced by Lactococcus lactis subsp. lactis. The final steps of lacticin 481 biosynthesis are proteolytic removal of an N-terminal leader sequence from the prepeptide LctA and export of the mature lantibiotic. Both proteolysis and secretion are performed by the dedicated ATP-binding cassette (ABC) transporter LctT. LctT belongs to the family of AMS (ABC transporter maturation and secretion) proteins whose prepeptide substrates share a conserved double-glycine type cleavage site. The in vitro activity of a lantibiotic protease has not yet been characterized. This study reports the purification and in vitro activity of the N-terminal protease domain of LctT (LctT150), and its use for the in vitro production of lacticin 481. The G(-2)A(-1) cleavage site and several other conserved amino acid residues in the leader peptide were targeted by site-directed mutagenesis to probe the substrate specificity of LctT as well as shed light upon the role of these conserved residues in lantibiotic biosynthesis. His 10 -LctT150 did not process most variants of the double glycine motif and processed mutants of Glu-8 only very slowly. Furthermore, incorporation of helix-breaking residues in the leader peptide resulted in greatly decreased proteolytic activity by His 10 -LctT150. On the other hand, His 10 -LctT150 accepted all peptides containing mutations in the propeptide or at non-conserved positions of LctA. In addition, the protease domain of LctT was investigated by site-directed mutagenesis of the conserved residues Cys12, His90, and Asp106. The proteolytic activities of the resulting mutant proteins are consistent with a cysteine protease.

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Effect of amino acid substitutions in conserved residues in the leader peptide on biosynthesis of the lantibiotic mutacin II

Cited by 38 publications

References 18 publications

Bovicin HJ50, a novel lantibiotic produced by Streptococcus bovis HJ50

Bovicin HJ50, a novel lantibiotic produced by Streptococcus bovis HJ50

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

In Vitro Reconstitution and Substrate Specificity of a Lantibiotic Protease

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