Proteases Involved in Leader Peptide Removal during RiPP Biosynthesis

Abstract

Ribosomally synthesized and post-translationally modified peptides (RiPPs) have received much attention in recent years because of their promising bioactivities and the portability of their biosynthetic pathways. Heterologous expression studies of RiPP biosynthetic enzymes identified by genome mining often leave a leader peptide on the final product to prevent toxicity to the host and to allow the attachment of a genetically encoded affinity purification tag. Removal of the leader peptide to produce the mature natural product is then carried out in vitro with either a commercial protease or a protease that fulfills this task in the producing organism. This review covers the advances in characterizing these latter cognate proteases from bacterial RiPPs and their utility as sequencedependent proteases. The strategies employed for leader peptide removal have been shown to be remarkably diverse. They include one-step removal by a single protease, two-step removal by two dedicated proteases, and endoproteinase activity followed by aminopeptidase activity by the same protease. Similarly, the localization of the proteolytic step varies from cytoplasmic cleavage to leader peptide removal during secretion to extracellular leader peptide removal. Finally, substrate recognition ranges from highly sequence specific with respect to the leader and/or modified core peptide to nonsequence specific mechanisms.

In Vitro Characterization of an O-Specific Glycosyltransferase Involved in Flagellin Glycosylation

“…This analysis identified several clusters of glycocin glycosyltransferases (Figure ). Glycosyltransferases such as SvGT, EnfC, and GccA, although functioning similarly, were not classified within the SunS/ThuS subfamily (InterPro: IPR026499). − Glycocin glycosyltransferases were thus further identified by their genomic proximity to C39 peptidases (InterPro: IPR005074), which cleave leader peptides from precursor glycocins . An SSN analysis also revealed clusters of glycosyltransferases within flagellar biosynthetic gene clusters containing flagellin genes (InterPro: IPR001492). , Sequence alignments and structure predictions using AlphaFold2 , suggest that these glycosyltransferases possess extended C-terminal α-helical domains with variable lengths (Figures , S1, and S2).…”

Biosynthesis of Macrocyclic Peptides with C-Terminal β-Amino-α-keto Acid Groups by Three Different Metalloenzymes

“…At present, it is unclear whether modified ApyA will be further processed by a leader peptidase. The BGC does not encode a protease, but akin to other RiPPs, the protease could be encoded elsewhere on the genome . Attempts to elicit the production of the final product by B. thailandensis E264 to provide information on proteolytic processing were unsuccessful.…”

Activity of Gut-Derived Nisin-like Lantibiotics against Human Gut Pathogens and Commensals

“…The leader peptide is recognized by modification enzymes that are usually encoded in the same biosynthetic gene cluster (BGC) to receive post-translational modifications on the core peptide. Subsequently, the leader peptide is removed by protease cleavage to yield the mature peptide with desired bioactivity. , Bioinformatic studies have found great potential for RiPP discovery from the microbiome . Recent advances in metagenomic sequencing and assembly have made vast amounts of environmental microbial genomes available for investigation.…”

“…Subsequently, the leader peptide is removed by protease cleavage to yield the mature peptide with desired bioactivity. 7 , 8 Bioinformatic studies have found great potential for RiPP discovery from the microbiome. 9 Recent advances in metagenomic sequencing and assembly have made vast amounts of environmental microbial genomes available for investigation.…”