lacticin-481 and Dehydration

The lantibiotics are a class of ribosomally synthesized and posttranslationally modified peptide antibiotics containing the thioether cross-links lanthionine (Lan) and 3-methyllanthionine (MeLan) and typically also the dehydroamino acids dehydroalanine (Dha) and (Z)-dehydrobutyrine (Dhb). The modifications are formed by dehydration of Ser/Thr residues to produce the Dha and Dhb structures, and subsequent conjugate additions of Cys residues onto the unsaturated amino acids to form thioether rings (Lan and MeLan). Because of their ribosomal origin, investigations of the substrate specificity of lantibiotic synthetases have typically focused on site-directed mutagenesis. With the in vitro reconstitution of lacticin 481 synthetase, its substrate specificity was explored in much greater detail by the incorporation of a series of nonproteinogenic Ser, Thr, and Cys analogues into a truncated prelacticin peptide LctA using a combination of solid-phase peptide synthesis (SPPS) and expressed protein ligation (EPL). The strategy described can be used for the growing number of ribosomally synthesized and posttranslationally modified natural products such as the microcins and patellamides.

Topics: Amino Acids; Analytic Sample Preparation Methods; Anti-Bacterial Agents; Bacterial Proteins; Bacteriocins; Cyclization; Cysteine; Dehydration; Gene Expression; Hydro-Lyases; Inteins; Ligases; Molecular Structure; Peptides; Protein Processing, Post-Translational; Recombinant Fusion Proteins; Serine; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Substrate Specificity; Threonine

Topics: Amino Acid Sequence; Bacteriocins; Dehydration; Enzymes; Molecular Conformation; Molecular Sequence Data; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stereoisomerism; Substrate Specificity

Lantibiotics are peptide antimicrobials containing the thioether-bridged amino acids lanthionine (Lan) and methyllanthionine (MeLan) and often the dehydrated residues dehydroalanine (Dha) and dehydrobutyrine (Dhb). While biologically advantageous, the incorporation of these residues into peptides is synthetically daunting, and their production in vivo is limited to peptides containing proteinogenic amino acids. The lacticin 481 synthetase LctM offers versatile control over the installation of dehydro amino acids and thioether rings into peptides. In vitro processing of semisynthetic substrates unrelated to the prelacticin 481 peptide demonstrated the broad substrate tolerance of LctM. Furthermore, a chemoenzymatic strategy was employed to generate novel thioether linkages by cyclization of peptidic substrates containing the nonproteinogenic cysteine analogs homocysteine and beta-homocysteine. These findings are promising with respect to the utility of LctM toward preparation of conformationally constrained peptide therapeutics.

Topics: Alanine; Amino Acid Sequence; Amino Acids; Bacteriocins; Cyclization; Dehydration; Enzyme Activation; Enzymes; Molecular Conformation; Molecular Sequence Data; Mutation; Peptides; Protein Engineering; Sensitivity and Specificity; Sulfides