Characterization of Histidine Functionalization and Its Timing in the Biosynthesis of Ribosomally Synthesized and Posttranslationally Modified Thioamitides

Abstract

Thioamitides are ribosomally synthesized and posttranslationally modified peptide (RiPP) natural products that hold great potential in anticancer drug development. Members in this RiPP family feature a thioamidated peptidyl chain conjugated with a macrocyclic ring system that contains two nonproteinogenic residues, 2-aminovinyl-cysteine (AviCys) and β-hydroxy-N,Ndimethyl-L-histidine (hdmHis). Focusing on the hdmHis residue that is unique to thioamitides, we report the enzymatic process for His functionalization and, more importantly, the timing of its related reactions with the other posttranslational modifications (PTMs) involved in thioamitide biosynthesis. His functionalization involves the activities of an S-adenosyl-L-methionine-dependent protein and a 2-oxoglutarate-Fe(II) monooxygenase for His bis-N-dimethylation and subsequent β-hydroxylation in a highly ordered manner. This process relies on the leader peptide sequence of the precursor peptide and on the establishment of the AviCyscontaining, C-terminal macrocyclic ring system in particular. In contrast, prior peptide thioamidation is not required. Knowledge gained from the catalytic logic, specificity, and compatibility of His functionalization greatly furthers our understanding of the process through which nature develops thioamitides from a ribosomally synthesized peptide precursor.

Discovery and biosynthesis of tricyclic copper-binding ribosomal peptides containing histidine-to-butyrine crosslinks

“…Common post-translational modifications of histidine residues, including methylation and phosphorylation, often rely on the nucleophilicity of the imidazole side-chain and a highly activated cofactor, such as S-adenosyl-L-methionine (SAM) and ATP [55][56][57] . Probably due to the lack of electrophilicity in proteinogenic amino acids, enzymatic side-chain crosslinking in peptides and proteins rarely utilize His residues as nucleophiles.…”

Non-modular Fatty Acid Synthases Yield Unique Acylation in Ribosomal Peptides

“…Alignment of these precursor peptides manifests a conserved TxxxHC motif, commonly found in thioviridamide-like thioamitides (Supplementary Table 4). We therefore hypothesized that the threonine and cysteine would form the AviMeCys moiety, and the histidine would be transformed into β-hydroxy- N,N -dimethyl- L -histidine (hdmHis) through methylation and hydroxylation by LpvM1 and LpvK, respectively 28, 29 (Supplementary Fig. 1).…”

“…8). The hdmHis was supported by the formation of 4-formyl-1,3-dimethyl-1 H -imidazolium fragment through a retro aldol reaction 24, 28, 31 . We also assigned a series of y-ions to the precursor peptide, with molecular weight differences implicating serine conversion into alanine and tyrosine glycosylation.…”

“…We next investigated the LpvE tolerance to the PTMs in the macrocyclic region. The hdmHis moiety exerts little impact on acylation, as acylated products consisting of unmodified and dimethylated histidine were observed upon disruption of LpvM1 and LpvK, respectively 28, 29 (Supplementary Fig. 46).…”