Substrate Recognition and Specificity of the NisB Protein, the Lantibiotic Dehydratase Involved in Nisin Biosynthesis

Mavaro, Antonino; Abts, André; Bakkes, Patrick J.; Moll, Gert N.; Driessen, Arnold J. M.; Smits, Sander H. J.; Schmitt, Lutz

doi:10.1074/jbc.m111.263210

Cited by 58 publications

(98 citation statements)

References 41 publications

(76 reference statements)

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“…Omitting the gel filtration step also did not provide active enzyme, and dehydrated peptide could not be detected after the addition of His 6 -NisA to purified His 6 -NisB in the presence of crude cell extracts of the E. coli cells that produced His 6 -NisB and fully dehydrated His 6 -NisA. These results are similar to the previously reported unsuccessful attempts to achieve in vitro activity of LanB enzymes (11)(12)(13)(14)(15).…”

Section: Resultssupporting

confidence: 81%

“…NisB was initially isolated from membrane vesicles of the nisin producer L. lactis in 1992 (11) and recently also purified in soluble form (15), but reconstitution of its activity in vitro has remained elusive. In this study, NisB activity was successfully reconstituted and required the membrane fraction of cell extract, ATP, Mg 2+ , and glutamate.…”

Section: Discussionmentioning

confidence: 99%

“…1B), and genetic and bioengineering studies have shown that the LanB for nisin biosynthesis (NisB) carries out the dehydration reaction and does not need any other lanthipeptide biosynthetic enzymes for activity (9,10). However, despite considerable effort in multiple laboratories, reconstitution of the activity of LanB dehydratases has been unsuccessful (11)(12)(13)(14)(15), and hence, no information is available about their mechanism of catalysis. The LanB dehydratases are 115-120 kDa proteins that do not share homology with the dehydratases from classes II-IV lanthipeptides or other proteins in the databases.…”

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confidence: 99%

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In vitro activity of the nisin dehydratase NisB

Garg¹,

Salazar-Ocampo

Donk

2013

Proc. Natl. Acad. Sci. U.S.A.

110

167

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The biosynthesis of several classes of ribosomally synthesized and posttranslationally modified peptides involves dehydration of serine and threonine residues. For class I lantibiotics, thiopeptides, and goadsporin, this dehydration is catalyzed by lanthionine biosynthetic enzyme B (LanB) or LanB-like proteins. Although LanB proteins have been studied since 1992, in vitro reconstitution of their dehydration activity has been elusive. We show here the in vitro activity of the dehydratase involved in the biosynthesis of the food preservative nisin (NisB). In vitro, NisB dehydrated its substrate peptide NisA eight times in the presence of glutamate, ATP, Mg 2+ , and the ribosomal/ membrane fraction of bacterial cell extract. Mutation of 23 highly conserved residues of NisB identified a number of amino acids that are essential for dehydration activity. In addition, these mutagenesis studies identified three mutants, R786A, R826A, and H961A, that result in multiple glutamylations of the NisA substrate. Glutamylation was observed during both Escherichia coli coexpression of NisA with these mutants and in vitro assays. Treatment of the glutamylated substrate with WT NisB results in dehydrated NisA, suggesting that the glutamylated peptide is an intermediate in dehydration. Collectively, these studies suggest that dehydration involves glutamylation of the side chains of Ser and Thr followed by elimination. The latter step has precedent in the virginiamycin resistance protein virginiamycin B lyase. These studies will facilitate investigation of other LanB proteins involved in the biosynthesis of lantibiotics, thiopeptides, and goadsporin.

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Section: Resultssupporting

confidence: 81%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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In vitro activity of the nisin dehydratase NisB

Garg¹,

Salazar-Ocampo

Donk

2013

Proc. Natl. Acad. Sci. U.S.A.

110

167

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“…In the case of the lantibiotic nisin, the precursor peptide NisA contains a conserved motif (F-D/N-L-N/D) within the leader peptide, which is essential for substrate recognition by the PTM enzyme NisB (11,(34)(35)(36). Mutagenesis of the FNLD motif within NisA dramatically decreased the binding to the dehydratase NisB (35,36).…”

Section: Discussionmentioning

confidence: 99%

Mutagenesis of NosM Leader Peptide Reveals Important Elements in Nosiheptide Biosynthesis

Jin

Xue

et al. 2017

Appl Environ Microbiol

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Nosiheptide, a typical member of the ribosomally synthesized and posttranslationally modified peptides (RiPPs), exhibits potent activity against multidrugresistant Gram-positive bacterial pathogens. The precursor peptide of nosiheptide (NosM) is comprised of a leader peptide with 37 amino acids and a core peptide containing 13 amino acids. To pinpoint elements in the leader peptide that are essential for nosiheptide biosynthesis, a collection of mutants with unique sequence features, including N-and C-terminal motifs, peptide length, and specific sites in the leader peptide, was generated by mutagenesis in vivo. The effects of various mutants on nosiheptide biosynthesis were evaluated. In addition to the necessity of a conserved motif LEIS box, native length and the N-terminal 12 amino acid residues were indispensable, and single-site substitutions of these 12 amino acid residues resulted in changes ranging from a greater-than-5-fold decrease to a 2-fold increase of nosiheptide production, depending on the sites and substituted residues. Moreover, although the C-terminal motif is not conservative, significant effects of this portion on nosiheptide production were also evident. Taken together, the present results further highlight the importance of the leader peptide in nosiheptide biosynthesis, and provide new insights into the diversity and specificity of leader peptides in the biosynthesis of various RiPPs.IMPORTANCE As a representative thiopeptide, nosiheptide exhibits excellent antibacterial activity. Although the biosynthetic gene cluster and several modification steps have been revealed, the presence and roles of the leader peptide within the precursor peptide of the nosiheptide gene cluster remain elusive. Thus, identification of specific elements in the leader peptide can significantly facilitate the genetic manipulation of the gene cluster for increasing nosiheptide production or generating diverse analogues. Given the complexity of the biosynthetic process, the instability of the leader peptide, and the unavailability of intermediates, cocrystallization of intermediates, leader peptide, and modification enzymes is currently not feasible. Therefore, a mutagenesis approach was used to construct a series of leader peptide mutants to uncover a number of crucial and characteristic elements affecting nosiheptide biosynthesis, which moves a considerable distance toward a thorough understanding of the biosynthetic machinery for thiopeptides.

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“…This literature search revealed the new buffer, illustrating that not every step towards a protein structure determination must be a trial-and-error process. Another example for the influence of the buffer composition was published bei Mavaro et al (Mavaro, Abts et al 2011). Instead of the buffer agent, the ionic strength of the buffer was the crucial determinant.…”

Section: Invisible Aggregationsmentioning

confidence: 99%