Mechanistic Studies on Dehydration in Class V Lanthipeptides

Abstract

Lanthipeptides are ribosomally synthesized and post-translationally modified peptides characterized by lanthionine (Lan) and/or methyllanthionine (MeLan) residues. Four classes of enzymes have been identified to install these structures in a substrate peptide. Recently, a novel class of lanthipeptides was discovered that lack genes for known class I–IV lanthionine synthases in their biosynthetic gene cluster (BGC). In this study, the dehydration of Ser/Thr during the biosynthesis of the class V lanthipeptide cacaoidin was reconstituted in vitro . The aminoglycoside phosphotransferase-like enzyme CaoK iteratively phosphorylates Ser/Thr residues on the precursor peptide CaoA, followed by phosphate elimination catalyzed by the HopA1 effector-like protein CaoY to achieve eight successive dehydrations. CaoY shows sequence similarity to the OspF family proteins and the lyase domains of class III/IV lanthionine synthetases, and mutagenesis studies identified residues that are critical for catalysis. An AlphaFold prediction of the structure of the dehydration enzyme complex engaged with its substrate suggests the importance of hydrophobic interactions between the CaoA leader peptide and CaoK in enzyme–substrate recognition. This model is supported by site-directed mutagenesis studies.

Genome Mining and Discovery of Imiditides, a Novel Family of RiPPs with a Class-defining Aspartimide Modification

“…The putative precursor peptide can be readily tested to see whether they can serve a substrate for nearby encoded putative RiPP enzymes to high accuracy when compared to experimental results based on previous studies. 35 Additionally, AlphaFold can also help indicate which reaction happens first when several enzymes are involved in the biosynthetic gene cluster (Figure S21). 17,35…”

“…35 Additionally, AlphaFold can also help indicate which reaction happens first when several enzymes are involved in the biosynthetic gene cluster (Figure S21). 17,35…”

Improved production of class I lanthipeptides in Escherichia coli

“…The five classes of lanthipeptides differ in the mechanisms of dehydration and cyclization. 3,15,19 Class I lanthipeptide biosynthesis features a dehydratase LanB that activates and eliminates the side-chain hydroxyl groups of Ser and Thr residues, and a cyclase LanC that forms Lan or MeLan (Fig. 1A).…”

Using LanM Enzymes to Modify Glucagon-Like Peptides 1 and 2 inE.coli

“…Some lanthipeptides are found with D-amino acids that are formed by hydrogenation of Dha and/or Dhb resulting in D-Ala and D-amino butyric acid, respectively. The enzymes that are responsible for the reduction reactions are called dehydrogenases and are collectively termed LanJ [15, 16]. The enzyme used in this study is called NpnJ, and is known to have a promiscuous substrate specificity in terms of peptide sequence, but specific to the reduction of Dha into D-Ala [17].…”