Rapid Determination of the Amino Acid Configuration of Xenotetrapeptide

Abstract

An E. coli strain with deletions in five transaminases (ΔaspC ΔilvE ΔtyrB ΔavtA ΔybfQ) was constructed to be unable to degrade several amino acids. This strain was used as an expression host for the analysis of the amino acid configuration of nonribosomally synthesized peptides, including the novel peptide "xenotetrapeptide" from Xenorhabdus nematophila, by using a combination of labeling experiments and mass spectrometry. Additionally, the number of D-amino acids in the produced peptide was assigned following simple cultivation of the expression strain in D2 O.

An Orthogonal D2O‐Based Induction System that Provides Insights into d‐Amino Acid Pattern Formation by Radical S‐Adenosylmethionine Peptide Epimerases

“…[5,7] Forarange of deuterated amino acids incorporated into expressed cores, we observed selective single deuterium-hydrogen exchange at the corresponding epimerized position. [5,7] Forarange of deuterated amino acids incorporated into expressed cores, we observed selective single deuterium-hydrogen exchange at the corresponding epimerized position.…”

Role of Secondary Metabolites in Establishment of the Mutualistic Partnership between Xenorhabdus nematophila and the Entomopathogenic Nematode Steinernema carpocapsae

“…These findings suggest that ngrA-dependent antimicrobials besides Xcn and compound F were produced at sufficient levels during preincubation to eliminate S. saprophyticus. X. nematophila produces several NRPS-derived compounds that possess antibiotic activity, including xenematide (33,34), PAX peptides (29,30), and compound C (present study), as well as other NRPS-derived compounds, such as xenotetrapeptide (35), whose activities have yet to be characterized (see Table S1 in the supplemental material). Whether these or other antimicrobial compounds are produced in Grace's medium and either individually or additively suppress the growth of sensitive competitors remains to be determined.…”

Introduction of d‐Amino Acids in Minimalistic Peptide Substrates by an S‐Adenosyl‐l‐Methionine Radical Epimerase

“…To pinpoint the exact OspA hydrogen abstracted during catalysis,al abeled substrate was generated by heterologous expression in minimal medium, supplemented with [a-D 1 ]-lisoleucine and [a-D 1 ]-l-valine,i nt he transaminase knockout strain E. coli Dpenta. [17] This selectively labeled precursor protein was reacted with OspD.HPLC-MS showed loss of the a-deuterium at Ile4 ( Figure 3A)and Va l13 ( Figure 3B), with ac oncomitant gain of deuterium by the AdoMet cleavage product dAdoC to give dAdoD ( Figure 3C and Supporting Information, Figure S4). In an intermolecular competition assay,t he overall rate of epimerization was seemingly unaffected by the deuterium label;y et, the dAdoH/dAdoD product ratio had negative slope (Supporting Information, Figure S5).…”

“…[17] This selectively labeled precursor protein was reacted with OspD.HPLC-MS showed loss of the a-deuterium at Ile4 ( Figure 3A)and Va l13 ( Figure 3B), with ac oncomitant gain of deuterium by the AdoMet cleavage product dAdoC to give dAdoD ( Figure 3C and Supporting Information, Figure S4). [17] This selectively labeled precursor protein was reacted with OspD.HPLC-MS showed loss of the a-deuterium at Ile4 ( Figure 3A)and Va l13 ( Figure 3B), with ac oncomitant gain of deuterium by the AdoMet cleavage product dAdoC to give dAdoD ( Figure 3C and Supporting Information, Figure S4).…”