Investigation of the Substrate Specificity of Lacticin 481 Synthetase by Using Nonproteinogenic Amino Acids

Abstract

One enzyme, many substrates. The substrate specificity of a lantibiotic biosynthetic enzyme, lacticin 481 synthetase, was probed by using synthetic prepeptides containing a variety of nonproteinogenic amino acids, including unnatural α‐amino acids, β‐amino acids, D‐amino acids, and peptoids. 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 β‐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.