Promiscuous Installation of d-Amino Acids in Gene-Encoded Peptides

Abstract

d-Amino acids can have major effects on the structure, proteolytic stability, and bioactivity of peptides. Proteusin radical S-adenosyl methionine epimerases regioselectively install such residues in ribosomal peptides to generate peptides with the largest number of d-residues currently known in biomolecules. To study their utility in synthetic biology, we investigated the substrate tolerance and substrate–product relationships of the cyanobacterial model epimerase OspD using libraries of point mutants as well as distinct extended peptides that were fused to an N-terminal leader sequence. OspD was found to exhibit exceptional substrate promiscuity in E. coli, accepting 15 different amino acids and converting peptides with a broad range of compositions, secondary structures, and polarities. Diverse single and multiple epimerization patterns were identified that were dictated by the peptide sequence. The data suggest major potential in creating genetically encoded products previously inaccessible by synthetic biology.

Leveraging Substrate Promiscuity of a Radical S-Adenosyl-L-methionine RiPP Maturase toward Intramolecular Peptide Cross-Linking Applications

“…While there have been several previous reports suggesting that RS RiPP maturases have varying levels of promiscuity, ,− the range of substrates that are processed by PapB, which include not just modified donor and acceptor residues and sequence context, but also the ability to accept unnatural amino acids, foreshadows a much larger substrate scope. We should point out that perhaps our ability to observe this expanded range of reactivity is fortuitous and related to access to a highly active protein.…”

Uncovering Novel Peptide Chemistry from Bacterial Natural Products

“…when fused to its cognate substrate leader sequence. [16] Proteusin epimerases therefore possess remarkable potential as synthetic tools for introducing peptide-stabilizing residues into therapeutic peptides.…”

“…[15] Proteusin epimerases displayed astonishing substrate promiscuity regarding epimerized amino acid residues and accepted core sequences. [15,16] Orthologs also produced similar epimerization patterns when supplied with the same substrate, highlighting stringent regioselectivity for these enzymes and suggesting that epimerization patterns are dictated, to an extent, by the core peptide sequence. The cyanobacterial epimerase homolog OspD (see below) was even capable of modifying nonnative peptide sequences from medically relevant targets including glucagon, GnRH, human amyloid β-peptide and protegrin-1 pacities in the 21 st century and the development of tools for the identification of these pathways, the RiPP class has seen a significant expansion that has unveiled a remarkable diversity of novel PTMs.…”

Emulating nonribosomal peptides with ribosomal biosynthetic strategies

“…45,47,48 However, d -amino acid patterns installed by a rSAM epimerase vary greatly depending on the core sequence and it is not yet possible to predict the product stereochemistry. 42…”

“…The rSAM peptide epimerase OspD from the proteusin landornamide pathway has been shown to install d -amino acids in highly diverse unnatural core sequences at many different proteinogenic residues and in different patterns (see Section 2.1 d -Amino acids). 42 Core-swap experiments using chimeric precursors as substrates for various OspD-like proteusin epimerases suggested that core elements rather than epimerase identity largely direct the regiospecificity of modifications to control the pattern of installed d -amino acids. 43 However, the core peptide elements that direct the site(s) of epimerisation are not yet defined and are therefore difficult to predict at this time.…”