Translation of Diverse Aramid- and 1,3-Dicarbonyl-peptides by Wild Type Ribosomes in Vitro

Abstract

Here, we report that wild type Escherichia coli ribosomes accept and elongate precharged initiator tRNAs acylated with multiple benzoic acids, including aramid precursors, as well as malonyl (1,3-dicarbonyl) substrates to generate a diverse set of aramid-peptide and polyketide-peptide hybrid molecules. This work expands the scope of ribozyme- and ribosome-catalyzed chemical transformations, provides a starting point for in vivo translation engineering efforts, and offers an alternative strategy for the biosynthesis of polyketide-peptide natural products.

Figure 1

Simple aminobenzoic acid cyanomethyl esters are poor substrates for the eFx ribozyme. (A) Protocol used to detect acylation of microhelix (MH) or tRNA by cyanomethyl esters 1–3. (B) Acid-urea gel-shift analysis of MH acylation by cyanomethyl esters 1–3 in the presence of ribozyme eFx. Yield was estimated by UV densitometry. (C) LC-HRMS analysis of MH acylation reactions after RNase A digestion. Adenine nucleosides acylated on the 2′ or 3′ hydroxyl of the 3′ terminal ribose of MH could be detected in eFx-promoted reactions of the cyanomethyl ester of l-phenylalanine (Phe) and aminobenzoic acid esters 1 and 2; trace levels were detected in reactions containing 3. These products were not observed in analogous reactions containing m-aminobenzoic acid (compound C).

Figure 2

Initiator tRNA (fMetT) acylated with o-aminobenzoic acid can initiate translation within the PTC of wild type E. coli ribosomes. (A) Protocol used to evaluate whether an initiator tRNA (fMetT) acylated with o- (prepared using isatoic anhydride) or m-aminobenzoic acid (prepared using eFx) (AN-tRNA) could support translation in vitro. (B) LC-HRMS analysis of reaction products showing DNA template-dependent translation of a polypeptide whose mass corresponds to that of o-AN-VFDYKDDDDK (o-AN-VF-FLAG). No such polypeptide is observed in the absence of DNA template or in the presence of l-methionine. LC-HRMS analysis of an analogous β-Phe-containing polypeptide is shown for comparison.

Figure 3

Probing structure–activity relationships for cyanomethyl esters of substituted benzoic acids in eFx-promoted acylation reactions. (A) Substituted benzoic acid cyanomethyl esters studied herein. (B) Acid-urea gel-shift analysis of MH acylation by cyanomethyl esters 6 and 8–15 in the presence of ribozyme eFx. Yield was estimated by UV densitometry. (C) LC-HRMS analysis of MH acylation reactions containing cyanomethyl esters 6 and 8–15 after RNase A digestion. Exact masses are reported in Table S2.

Figure 4

Initiator tRNAs acylated with diverse benzoic acids are accommodated in the ribosomal P-site and are elongated into AR-VF-FLAG polypeptides. LC-HRMS analysis of reaction products whose masses correspond to AR-VFDYKDDDDK (AR-VF-FLAG) polypeptides containing diverse substituted benzoic acid monomers.

Figure 5

Wild type E. coli ribosomes support the biosynthesis of polyketide-peptide hybrid molecules. (A) Malonic esters 19–23 evaluated as substrates for eFx or dFx. (B) Acid-urea gel-shift analysis of MH acylation by esters 19–23 in the presence of eFx (19, 22) or dFx (20, 21,23). Yield was estimated by UV densitometry. (C) LC-HRMS analysis of MH acylation reactions containing esters 19–23 after RNase A digestion. (D) LC-HRMS analysis of reaction products whose masses corresponds to Mal-VFDYKDDDDK (Mal-VF-FLAG) polypeptides containing methyl and nitrobenzyl malonates 23 and 22. ND = not determined due to lack of separation from unacylated microhelix. Exact masses are reported in Table S2.