Identification of the peptide epimerase MslH responsible for d-amino acid introduction at the C-terminus of ribosomal peptides

Abstract

The biosynthesis of d-tryptophan containing lasso peptide MS-271 involves the epimerization of a ribosomal peptide MslA catalyzed by a novel class of metal- and cofactor-independent peptide epimerase MslH.

Recent Advances and Perspectives on Expanding the Chemical Diversity of Lasso Peptides

“…Concurrently, a genome mining study discovered another D-amino acid-containing lasso peptide, specialicin, encoded by a similarly organized gene cluster (Kaweewan et al, 2018). Followup in vitro experiments on the core peptide and full-length precursor peptide demonstrated the mslH gene product to be responsible for introducing the D-amino acid (Feng et al, 2021). Similar to the reported lasso peptide methyltransferase (StspM), kinases (ThcoK and SyanK), and iron/2-oxoglutarate-dependent hydroxylase (CanE), MslH specifically recognizes the linear precursor peptide but not the folded lasso peptide, a feature which renders these enzymes useful for peptide engineering.…”

Unusual Post-Translational Modifications in the Biosynthesis of Lasso Peptides

“…The auxiliary PTMs such as phosphorylation, methylation, hydroxylation, and epimerization recognize the linear precursors ahead of the removal of leader peptides [25,26,31], of which the hydroxylation and epimerization have been confirmed to be promoted by the B1 proteins [40,51]. The possibilities of the promotion by B1 proteins for phosphorylation and methylation cannot be ruled out.…”

“…Homologous genes were also identified in other BGCs such as specialicin [14], aborycin [15], humidimycin [16], and poly-γ-glutamic acid (PGA) [50], a biopolymer that comprises D-and L-glutamic acid connected via amide bonds, but were absent in the BGCs of non-D-Trp containing class I lasso peptides such as sviceucin [17] and arcumycin [18] (Figure 2b). The function of MslH was further validated for the epimerization of the C-terminal L-Trp in vitro [51]. The full-length precursor MslA is the most favorite substrate for MslH, as the compared reaction with leaderless core peptide only produced a minor amount of D-Trp.…”

“…Since the C-terminal L-Trp derivative has never been detected in the MS-271 producer, the following MslB2 and MslC maturation processes probably recognize epi-MslA as the sole substrate and drive the equilibrium to D-Trp containing precursor peptide (Figure 7b). Furthermore, MslH could epimerize other aromatic residues such as W21F and W21Y at considerable levels, and chimeric substrates with the sviceucin N-terminal core peptide sequence and the C-terminal "CFW" (Figure 2b), displaying a broad substrate tolerance [51].…”

“…For instance, the single radical S-adenosylmethionine (SAM) peptide epimerase PoyD introduces up to 18 D-amino acids in the biosynthesis of polytheonamides [52], another radical SAM epimerase YydG epimerizes the formation of a D-Val and D-allo-Ile residues in the biosynthesis of the epipeptide YydF [53]. Additionally, D-Ala and D-amino butyric acid (D-Abu) residues are introduced into lanthipeptides by the hydrogenation of 2,3didehydroalanine (Dha, dehydrated Ser) and 2,3-didehydrobutyrine (Dhb, dehydrated Thr) via different oxidoreductases, including the zinc-dependent dehydrogenases termed The function of MslH was further validated for the epimerization of the C-terminal L-Trp in vitro [51]. The full-length precursor MslA is the most favorite substrate for MslH, as the compared reaction with leaderless core peptide only produced a minor amount of D-Trp.…”