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Lacticin 481 synthetase as a general serine/threonine kinase.

ABSTRACT: Methods that introduce posttranslational modifications in a general, mild, and non-sequence-specific manner using biologically produced peptides have great utility for investigation of the functions of these modifications. In this study, the substrate promiscuity of a lantibiotic synthetase was exploited for the preparation of phosphopeptides, glycopeptides, and peptides containing analogs of methylated or acetylated lysine residues. Peptides attached to the C-terminus of the leader peptide of the lacticin 481 precursor peptide were phosphorylated on serine residues in a wide variety of sequence contexts by the R399M and T405A mutants of lacticin 481 synthetase (LctM). Serine residues located as many as 30 amino acids C-terminal to the leader peptide were phosphorylated. Wild-type LctM was shown to dehydrate these peptides to generate dehydroalanine-containing products that can be conveniently modified with external nucleophiles including thiosaccharides, 2-(dimethylamino)ethanethiol, and N-acetyl cysteamine, resulting in mimics of O-linked glycopeptides and acetylated and methylated lysines.

SUBMITTER: You YO

PROVIDER: S-EPMC2709986 | biostudies-literature |

REPOSITORIES: biostudies-literature

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On the regioselectivity of thioether formation by lacticin 481 synthetase.

Project description:Lantibiotic synthetases generate intramolecular thioether cross-links within peptides through the Michael-type addition of cysteines onto dehydroamino acids originating from Ser and Thr. Presented here is an assay that readily distinguishes between enzymatic and nonenzymatic formation of these cross-links. The results demonstrate unequivocally that lacticin 481 synthetase can generate non-native thioether cross-links.

| S-EPMC2517117 | biostudies-literature

The dehydratase activity of lacticin 481 synthetase is highly processive.

Project description:Lacticin 481 synthetase (LctM) is a bifunctional enzyme that undertakes dehydration and cyclization in the structural region of the pre-lacticin peptide (LctA) to introduce three thioether rings and one dehydrobutyrine residue. The order and timing of these events has been investigated employing high-resolution ESI-FTMS-based tandem MS/MS techniques and chemical derivatization. LctM demonstrates highly processive behavior as seen by MS analysis of the reaction course of dehydration. Furthermore, cyclization is not tightly coupled to dehydration and follows at a later stage of the enzymatic reaction.

| S-EPMC2532561 | biostudies-literature

Investigation of the substrate specificity of lacticin 481 synthetase by using nonproteinogenic amino acids.

Project description:Lantibiotics are peptide antimicrobial compounds that are characterized by the thioether-bridged amino acids lanthionine and methyllanthionine. For lacticin 481, these structures are installed in a two-step post-translational modification process by a bifunctional enzyme, lacticin 481 synthetase (LctM). LctM catalyzes the dehydration of Ser and Thr residues to generate dehydroalanine or dehydrobutyrine, respectively, and the subsequent intramolecular regio- and stereospecific Michael-type addition of cysteines onto the dehydroamino acids. In this study, semisynthetic substrates containing nonproteinogenic amino acids were prepared by expressed protein ligation and [3+2]-cycloaddition of azide and alkyne-functionalized peptides. LctM demonstrated broad substrate specificity toward substrates containing beta-amino acids, D-amino acids, and N-alkyl amino acids (peptoids) in certain regions of its peptide substrate. These findings showcase its promise for use in lantibiotic and peptide-engineering applications, whereby nonproteinogenic amino acids might impart improved stability or modulated biological activities. Furthermore, LctM permitted the incorporation of an alkyne-containing amino acid that can be utilized for the site-selective modification of mature lantibiotics and used in target identification.

| S-EPMC2737179 | biostudies-literature

Mechanistic investigations of the dehydration reaction of lacticin 481 synthetase using site-directed mutagenesis.

Project description:Lantibiotic synthetases catalyze the dehydration of Ser and Thr residues in their peptide substrates to dehydroalanine (Dha) and dehydrobutyrine (Dhb), respectively, followed by the conjugate addition of Cys residues to the Dha and Dhb residues to generate the thioether cross-links lanthionine and methyllanthionine, respectively. In this study ten conserved residues were mutated in the dehydratase domain of the best characterized family member, lacticin 481 synthetase (LctM). Mutation of His244 and Tyr408 did not affect dehydration activity with the LctA substrate whereas mutation of Asn247, Glu261, and Glu446 considerably slowed down dehydration and resulted in incomplete conversion. Mutation of Lys159 slowed down both steps of the net dehydration: phosphorylation of Ser/Thr residues and the subsequent phosphate elimination step to form the dehydro amino acids. Mutation of Arg399 to Met or Leu resulted in mutants that had phosphorylation activity but displayed greatly decreased phosphate elimination activity. The Arg399Lys mutant retained both activities, however. Similarly, the Thr405Ala mutant phosphorylated the LctA substrate but had compromised elimination activity. Finally, mutation of Asp242 or Asp259 to Asn led to mutant enzymes that lacked detectable dehydration activity. Whereas the Asp242Asn mutant retained phosphate elimination activity, the Asp259Asn mutant was not able to eliminate phosphate from a phosphorylated substrate peptide. A model is presented that accounts for the observed phenotypes of these mutant enzymes.

| S-EPMC2517115 | biostudies-literature

The leader peptide is not required for post-translational modification by lacticin 481 synthetase.

Project description: Not available

| S-EPMC2492579 | biostudies-literature

Mutants of the zinc ligands of lacticin 481 synthetase retain dehydration activity but have impaired cyclization activity.

Project description:Lantibiotics are ribosomally synthesized and post-translationally modified peptide antibiotics. The modifications involve dehydration of Ser and Thr residues to generate dehydroalanines and dehydrobutyrines, followed by intramolecular attack of cysteines onto the newly formed dehydro amino acids to produce cyclic thioethers. LctM performs both processes during the biosynthesis of lacticin 481. Mutation of the zinc ligands Cys781 and Cys836 to alanine did not affect the dehydration activity of LctM. However, these mutations compromised cyclization activity when investigated with full length or truncated peptide substrates. Mutation of His725, another residue that is fully conserved in lantibiotic cyclases, to Asn resulted in a protein that still catalyzed dehydration of the substrate peptide and also retained cyclization activity, but at a decreased level compared to that of the wild type enzyme. Collectively, these results show that the C-terminal domain of LctM is responsible for cyclization, that the zinc ligands are critical for cyclization, and that dehydration takes place independently from the cyclization activity. Furthermore, these mutant proteins are excellent dehydratases and provide useful tools to investigate the dehydration activity as well as generate dehydrated peptides for study of the cyclization reaction by wild type LctM.

| S-EPMC2517114 | biostudies-literature

Serine/Threonine Protein Kinase STK16.

Project description:STK16 (Ser/Thr kinase 16, also known as Krct/PKL12/MPSK1/TSF-1) is a myristoylated and palmitoylated Ser/Thr protein kinase that is ubiquitously expressed and conserved among all eukaryotes. STK16 is distantly related to the other kinases and belongs to the NAK kinase family that has an atypical activation loop architecture. As a membrane-associated protein that is primarily localized to the Golgi, STK16 has been shown to participate in the TGF-β signaling pathway, TGN protein secretion and sorting, as well as cell cycle and Golgi assembly regulation. This review aims to provide a comprehensive summary of the progress made in recent research about STK16, ranging from its distribution, molecular characterization, post-translational modification (fatty acylation and phosphorylation), interactors (GlcNAcK/DRG1/MAL2/Actin/WDR1), and related functions. As a relatively underexplored kinase, more studies are encouraged to unravel its regulation mechanisms and cellular functions.

| S-EPMC6480182 | biostudies-literature

Altered serine/threonine kinase activity in schizophrenia.

Project description:Converging evidence implicates alterations in multiple signaling pathways in the etiology of schizophrenia. Previously, these studies were limited to the analysis of one or a few phosphoproteins at a time. Here, we use a novel kinase array platform to simultaneously investigate the convergence of multiple signaling cascades implicated in schizophrenia. This technology uses consensus peptide substrates to assess activity levels of a large number (>100) of serine/threonine protein kinases. 19 peptide substrates were differentially phosphorylated (>15% change) in the frontal cortex in schizophrenia. These peptide substrates were examined using Ingenuity Pathway Analysis to group them according to the functions and to identify processes most likely affected in schizophrenia. Pathway analysis placed 14 of the 19 peptides into cellular homeostatic pathways, 10 into pathways governing cytoskeletal organization, and 8 into pathways governing ion homeostasis. These data are the first to simultaneously investigate comprehensive changes in signaling cascades in a severe psychiatric disorder. The examination of kinase activity in signaling pathways may facilitate the identification of novel substrates for drug discovery and the development of safer and more effective pharmacological treatment for schizophrenia.

| S-EPMC4096904 | biostudies-literature

The importance of the leader sequence for directing lanthionine formation in lacticin 481.

Project description:Lantibiotics are post-translationally modified peptide antimicrobial agents that are synthesized with an N-terminal leader sequence and a C-terminal propeptide. Their maturation involves enzymatic dehydration of Ser and Thr residues in the precursor peptide to generate unsaturated amino acids, which react intramolecularly with nearby cysteines to form cyclic thioethers termed lanthionines and methyllanthionines. The role of the leader peptide in lantibiotic biosynthesis has been subject to much speculation. In this study, mutations of conserved residues in the leader sequence of the precursor peptide for lacticin 481 (LctA) did not inhibit dehydration and cyclization by lacticin 481 synthetase (LctM) showing that not one specific residue is essential for these transformations. These amino acids may therefore be conserved in the leader sequence of class II lantibiotics to direct other biosynthetic events, such as proteolysis of the leader peptide or transport of the active compound outside the cell. However, introduction of Pro residues into the leader peptide strongly affected the efficiency of dehydration, consistent with recognition of the secondary structure of the leader peptide by the synthetase. Furthermore, the presence of a hydrophobic residue at the position of Leu-7 appears important for enzymatic processing. Based on the data in this work and previous studies, a model for the interaction of LctM with LctA is proposed. The current study also showcases the ability to prepare other lantibiotics in the class II lacticin 481 family, including nukacin ISK-1, mutacin II, and ruminococcin A using the lacticin 481 synthetase. Surprisingly, a conserved Glu located in a ring that appears conserved in many class II lantibiotics, including those not belonging to the lacticin 481 subgroup, is not essential for antimicrobial activity of lacticin 481.

| S-EPMC2574844 | biostudies-literature

Chemical synthesis of the lantibiotic lacticin 481 reveals the importance of lanthionine stereochemistry.

Project description:Lantibiotics are a family of antibacterial peptide natural products characterized by the post-translational installation of the thioether-containing amino acids lanthionine and methyllanthionine. Until recently, only a single naturally occurring stereochemical configuration for each of these cross-links was known. The discovery of lantibiotics with alternative lanthionine and methyllanthionine stereochemistry has prompted an investigation of its importance to biological activity. Here, solid-supported chemical synthesis enabled the total synthesis of the lantibiotic lacticin 481 and analogues containing cross-links with non-native stereochemical configurations. Biological evaluation revealed that these alterations abolished the antibacterial activity in all of the analogues, revealing the critical importance of the enzymatically installed stereochemistry for the biological activity of lacticin 481.

| S-EPMC3736828 | biostudies-literature

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