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.

Developments and recent advancements in the field of endogenous amino acid selective bond forming reactions for bioconjugation

“…Several other approaches continue to appear and are designed to overpass this problematic. These are oxidative elimination of aryl-selenocysteine, 399,[404][405][406][407][408] utilising of lacticin synthetase, 409 transformation to selenocysteine thioethers. 410 1.3.9 Miscellaneous thiol-selective reagents.…”

An Improved Synthesis of the Antibiotic Dehydrophos

“…It should be noted that the introduction of a methylene group under such smooth conditions is also very rare in the case of the carboxylic acid counterparts. Indeed, the synthetic methodologies described in the literature for the preparation of peptides containing a dehydroalanine moiety comprise the elimination reaction of serine,, cysteine or selenocysteine residues.…”

Recent Developments in Neoglycopeptide Synthesis

“…Similarly, incorporation of an azido tag into the flagella of pathogenic bacteria has been performed on Campylobacter jejuni cells through cultivation in a motility agar containing an azido-functionalized precursor [145]. Peptides attached at the C-terminus of a leader peptide for recognition by lacticin synthetase mutants have been generated by expression in E. coli [146]. After incubation with lacticin synthetases, serine and threonine residues up to 30 amino acids distant from the leader peptide have been dehydrated.…”

“…After incubation with lacticin synthetases, serine and threonine residues up to 30 amino acids distant from the leader peptide have been dehydrated. The resulting dehydroalanines were subsequently glycosylated by addition of thio-sugars [146]. Expressed protein ligation has been used to introduce cysteine-containing molecules at the terminus of bacterially expressed proteins.…”