Detection of Modified Amino Acids in Lantibiotic Peptide Mutacin II by Chemical Derivatization and Electrospray Ionization–Mass Spectroscopic Analysis

Novák, Jan; Kirk, Marion; Caufield, Page W.; Barnes, Stephen; Morrison, Kelly; Baker, John

doi:10.1006/abio.1996.0181

Cited by 23 publications

(24 citation statements)

References 7 publications

(15 reference statements)

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“…To maintain high sensitivity, an external calibration was applied. Ethanethiol treatment (35) was applied to confirm posttranslational modification.…”

Section: Methodsmentioning

confidence: 99%

NisT, the Transporter of the Lantibiotic Nisin, Can Transport Fully Modified, Dehydrated, and Unmodified Prenisin and Fusions of the Leader Peptide with Non-lantibiotic Peptides

Kuipers¹,

Boef

Rink

et al. 2004

Journal of Biological Chemistry

150

214

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Lantibiotics are lanthionine-containing peptide antibiotics. Nisin, encoded by nisA, is a pentacyclic lantibiotic produced by some Lactococcus lactis strains. Its thioether rings are posttranslationally introduced by a membrane-bound enzyme complex. This complex is composed of three enzymes: NisB, which dehydrates serines and threonines; NisC, which couples these dehydrated residues to cysteines, thus forming thioether rings; and the transporter NisT. We followed the activity of various combinations of the nisin enzymes by measuring export of secreted peptides using antibodies against the leader peptide and mass spectroscopy for detection. L. lactis expressing the nisABTC genes efficiently produced fully posttranslationally modified prenisin. Strikingly, L. lactis expressing the nisBT genes could produce dehydrated prenisin without thioether rings and a dehydrated form of a non-lantibiotic peptide. In the absence of the biosynthetic NisBC enzymes, the NisT transporter was capable of excreting unmodified prenisin and fusions of the leader peptide with nonlantibiotic peptides. Our data show that NisT specifies a broad spectrum (poly)peptide transporter that can function either in conjunction with or independently from the biosynthetic genes. NisT secretes both unmodified and partially or fully posttranslationally modified forms of prenisin and non-lantibiotic peptides. These results open the way for efficient production of a wide range of peptides with increased stability or novel bioactivities.

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“…To maintain high sensitivity, an external calibration was applied. Ethanethiol treatment (35) was applied to confirm posttranslational modification.…”

Section: Methodsmentioning

confidence: 99%

NisT, the Transporter of the Lantibiotic Nisin, Can Transport Fully Modified, Dehydrated, and Unmodified Prenisin and Fusions of the Leader Peptide with Non-lantibiotic Peptides

Kuipers¹,

Boef

Rink

et al. 2004

Journal of Biological Chemistry

150

214

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“…To confirm that there were indeed six dehydrated residues in the mature mutacin I, we performed an ethanethiol modification of mutacin I under alkaline conditions. In this reaction, one molecule of ethanethiol could insert into the thioether bridge, resulting in a Sethylcysteine and a cysteine, or it could insert into the double bond of a dehydrated serine or threonine to form an S-ethyl- cysteine or ␤-methyl-S-ethylcysteine (20,23). Ethanethiol derivatization of lantibiotics has been used prior to sequencing of the other lantibiotics gallidermin and pep5 (20) and for determination of the number of dehydrated amino acid residues in mutacin II (23).…”

Section: Fig 1 (A)mentioning

confidence: 99%

“…The pellet fraction after 30% acetonitrile wash contained mostly the full-length modified mutacin I and the N-terminal fragment. The pellet fraction was found to have the sequence 23 . S-Ethylcysteine (SEC) was the product of ethanethiol insertion into the double bond of dehydrated serine or the thioether bridge in lanthionine.…”

Section: Fig 1 (A)mentioning

confidence: 99%

“…All lantibiotic loci also contain a set of immunity genes, which are responsible for self-protection of the producer strains (33). Moreover, expression of the lantibiotic genes is usually regulated either by a single transcription regulator (24,27) or by a two-component signal transduction system (5,15,16).Previously, our group reported isolation, biochemical, and genetic characterizations of mutacin II, produced by a group II strain of the oral bacterium Streptococcus mutans (3,22,23,27). Mutacin II belongs to subgroup AII in the lantibiotic family.…”

mentioning

confidence: 99%

“…Previously, our group reported isolation, biochemical, and genetic characterizations of mutacin II, produced by a group II strain of the oral bacterium Streptococcus mutans (3,22,23,27). Mutacin II belongs to subgroup AII in the lantibiotic family.…”

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confidence: 99%

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Purification and Biochemical Characterization of Mutacin I from the Group I Strain of Streptococcus mutans , CH43, and Genetic Analysis of Mutacin I Biosynthesis Genes

Chen

Caufield

2000

Appl Environ Microbiol

Self Cite

106

138

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Previously, we reported isolation and characterization of mutacin III and genetic analysis of mutacin III biosynthesis genes from the group III strain of Streptococcus mutans, UA787 (F. Qi, P. Chen, and P. W. Caufield, Appl. Environ. Microbiol. 65:3880-3887, 1999). During the same process of isolating the mutacin III structural gene, we also cloned the structural gene for mutacin I. In this report, we present purification and biochemical characterization of mutacin I from the group I strain CH43 and compare mutacin I and mutacin III biosynthesis genes. The mutacin I biosynthesis gene locus consists of 14 genes in the order mutR, -A, -A, -B, -C, -D, -P, -T, -F, -E, -G, orfX, orfY, orfZ. mutA is the structural gene for mutacin I, while mutA is not required for mutacin I activity. DNA and protein sequence analysis revealed that mutacins I and III are homologous to each other, possibly arising from a common ancestor. The mature mutacin I is 24 amino acids in size and has a molecular mass of 2,364 Da. Ethanethiol modification and peptide sequencing of mutacin I revealed that it contains six dehydrated serines, four of which are probably involved with thioether bridge formation. Comparison of the primary sequence of mutacin I with that of mutacin III and epidermin suggests that mutacin I likely has the same bridging pattern as epidermin.Lantibiotics are lanthionine-containing small-peptide antibiotics that are produced by gram-positive bacteria (11, 32). The lantibiotics are ribosomally synthesized and posttranslationally modified (34). The modification reactions include dehydration of serine and threonine residues and addition of thiol groups from cysteine residues to the double bond to form lanthionines and ␤-methyllanthionines, respectively. Some dehydrated serine or threonine residues may remain as such in the mature lantibiotic peptide.Based on the secondary structures, Jung assigned lantibiotics into two classes, types A (linear) and B (globular) (11). de Vos et al. (6) and Sahl and Bierbaum (31) further divided each class into subgroups according to their primary peptide sequences. Thus, subgroup AI contains the nisin-like lantibiotics, with nisin, subtilin, epidermin, and pep5 as the most thoroughly characterized members (1,7,8,12,38). Subgroup AII consists of lacticin 481, salivaricin, and variacin (10,25,26,30). The genes responsible for biosynthesis of lantibiotics are organized in operon-like structures. The biosynthesis locus of all members in the subgroup AI lantibiotics consists of lanA, the structural gene for the lantibiotic; lanB and lanC, the modifying enzyme genes for posttranslational modification of the preprolantibiotic; lanP, the protease gene for processing of the prelantibiotic; and lanT, the ABC transporter for secretion of the lantibiotic. In addition, epidermin and gallidermin have an extra gene, lanD, which is responsible for the C-terminal oxidative decarboxylation of the lantibiotic (17, 18). In comparison, subgroup AII lantibiotics have simpler genomic organizations. In subgroup AII, ...

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Chemistry and Structure

Jack

Bierbaum

Sahl

1998

Lantibiotics and Related Peptides

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Detection of Modified Amino Acids in Lantibiotic Peptide Mutacin II by Chemical Derivatization and Electrospray Ionization–Mass Spectroscopic Analysis

Cited by 23 publications

References 7 publications

NisT, the Transporter of the Lantibiotic Nisin, Can Transport Fully Modified, Dehydrated, and Unmodified Prenisin and Fusions of the Leader Peptide with Non-lantibiotic Peptides

NisT, the Transporter of the Lantibiotic Nisin, Can Transport Fully Modified, Dehydrated, and Unmodified Prenisin and Fusions of the Leader Peptide with Non-lantibiotic Peptides

Purification and Biochemical Characterization of Mutacin I from the Group I Strain of Streptococcus mutans , CH43, and Genetic Analysis of Mutacin I Biosynthesis Genes

Chemistry and Structure

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