Mechanistic Studies of Ser/Thr Dehydration Catalyzed by a Member of the LanL Lanthionine Synthetase Family

Goto, Yuki; Ökesli, Ayşe; Donk, Wilfred A. van der

doi:10.1021/bi101750r

Cited by 58 publications

(89 citation statements)

References 24 publications

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“…Interestingly, a similar mechanism of catalysis was identified recently in members of the bacterial LanL family of lanthionine synthetases that share sequence homology with OspF family members (Goto et al, 2011). This enzyme family is responsible for biosynthesis of lantibiotics, a class of peptide antibiotics, through a process that includes phosphorylation followed by dehydration of phosphothreonine and phosphoserine (Willey and van der Donk, 2007).…”

Section: Phosphorylationsupporting

confidence: 57%

What Pathogens Have Taught Us About Posttranslational Modifications

Salomon

Orth

2013

Cell Host & Microbe

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Pathogens use various mechanisms to manipulate host processes to promote infection. Decades of research on pathogens have revealed not only the molecular mechanisms that these microbes use to replicate and survive within host cells, but also seminal information on how host signaling machinery regulates cellular processes. Among these discoveries are mechanisms involving posttranslational modifications that alter the activity, localization, or interactions of the modified protein. Herein, we examine how pathogens have contributed to our basic understanding of three posttranslational modifications: phosphorylation, NMPylation, and ubiquitylation. Over the years, technologies, techniques and research tools have developed side by side with the study of pathogens, facilitating the discovery of protein modifications and furthering our understanding of how they contribute to both infection and cellular functions.

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Section: Phosphorylationsupporting

confidence: 57%

What Pathogens Have Taught Us About Posttranslational Modifications

Salomon

Orth

2013

Cell Host & Microbe

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“…(14) In contrast to other families of lanthipeptide dehydratases, which employ phosphorylation to activate the β-hydroxyl as a leaving group, NisB dehydrates the serine and threonine residues of the nisin precursor peptide NisA via a glutamylated intermediate, and the LanB-like dehydratases involved in thiopeptide maturation likely adopt a similar catalytic strategy. (35–37) The LanB dehydratases demonstrate a broad tolerance toward non-native peptide substrates and are flexible in their abilities to dehydrate serine and threonine residues introduced by precursor peptide engineering. (38-40) For example, the Lys12Ser and Lys12Thr mutants of NisA both led to the production of a mixture of nisin analogs either unmodified or dehydrated at the twelfth position, with dehydration of the threonine residue predominating.…”

Section: Resultsmentioning

confidence: 99%

Saturation Mutagenesis of TsrA Ala4 Unveils a Highly Mutable Residue of Thiostrepton A

Zhang

Kelly

2015

ACS Chem. Biol.

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Thiopeptides are posttranslationally processed macrocyclic peptide metabolites, characterized by extensive backbone and side chain modifications that include a six-membered nitrogeneous ring, thiazol(in)e/oxazol(in)e rings, and dehydrated amino acid residues. Thiostrepton A, one of the more structurally complex and well-studied thiopeptides, contains a second macrocycle bearing a quinaldic acid moiety. Antibacterial, antimalarial, and anticancer properties have been described for thiostrepton A and other thiopeptides, although the molecular details for binding the cellular target in each case are not fully elaborated. We previously demonstrated that a mutation of the TsrA core peptide, Ala4Gly, supported the successful production of the corresponding thiostrepton variant. To more thoroughly probe the thiostrepton biosynthetic machinery’s tolerance toward structural variation at the fourth position of the TsrA core peptide, we report here the saturation mutagenesis of this residue using a fosmid-dependent biosynthetic engineering method and the isolation of 16 thiostrepton analogs. Several types of side chain substitutions at the fourth position of TsrA, including those that introduce polar or branched, hydrophobic residues, are accepted, albeit with varied preferences. In contrast, proline and amino acid residues inherently charged at physiological pH are not well-tolerated at the queried site by the thiostrepton biosynthetic system. These newly generated thiostrepton analogs were assessed for their antibacterial activities and abilities to inhibit the proteolytic functions of the eukaryotic 20S proteasome. We demonstrate that the identity of the fourth amino acid residue in the thiostrepton scaffold is not critical for either ribosome or proteasome inhibition.

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“…On deprotonation of the α-proton of the glutamylated Ser or Thr residues, elimination of glutamate results in dehydroalanine/dehydrobutyrine residues. It is possible that glutamylation and elimination occur in different domains, which is also observed for class III and class IV lanthipeptides that contain separate kinase and lyase domains (7,28). If so, then the C terminus of NisB contains the likely lyase domain.…”

Section: Discussionmentioning

confidence: 95%

In vitro activity of the nisin dehydratase NisB

Garg¹,

Salazar-Ocampo

Donk

2013

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

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111

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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Mechanistic Studies of Ser/Thr Dehydration Catalyzed by a Member of the LanL Lanthionine Synthetase Family

Cited by 58 publications

References 24 publications

What Pathogens Have Taught Us About Posttranslational Modifications

What Pathogens Have Taught Us About Posttranslational Modifications

Saturation Mutagenesis of TsrA Ala4 Unveils a Highly Mutable Residue of Thiostrepton A

In vitro activity of the nisin dehydratase NisB

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