Lacticin 481 Synthetase as a General Serine/Threonine Kinase

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 <i>N</i>-acetyl cysteamine, resulting in mimics of O-linked glycopeptides and acetylated and methylated lysines

Distributive and Directional Behavior of Lantibiotic Synthetases Revealed by High-Resolution Tandem Mass Spectrometry

The lantibiotic synthetases LctM and HalM2 are bifunctional enzymes that catalyze both the dehydration of serine and threonine residues and the Michael-type additions of cysteine residues to the resulting dehydroamino acids in their substrate peptides. Using Fourier transform mass spectrometry to analyze these activities <i>in vitro</i>, the dehydration is shown to take place by a distributive mechanism, with build-up of intermediates observed in electrospray mass spectra. The cyclization activity of HalM2 was monitored through alkylation of free cysteines in intermediates, providing access to the regioselectivity of lanthionine ring formation using high-resolution tandem mass spectrometry. HalM2 is shown to catalyze the cyclization process in a largely N- to C-terminal directional fashion, forming a total of four lanthionine rings in its HalA2 substrate. These studies advance a model for lantibiotic production where substrate binding <i>via</i> an N-terminal leader results in dehydration and cyclization on similar time scales and with a high, though not strict, propensity for N-to-C directionality