Synthetic post‐translational modifications of elongation factor P using the ligase EpmA

Pfab, Miriam; Kielkowski, Pavel; Krafczyk, Ralph; Volkwein, Wolfram; Sieber, Stephan A.; Lassak, Jürgen; Jung, Kirsten

doi:10.1111/febs.15346

Cited by 7 publications

(4 citation statements)

References 64 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This cannot necessarily be expected, as the E. coli endogenous EF-P significantly differs from its pseudomonal counterpart 25 : although both proteins alleviate ribosome stalling at polyproline stretches 6 , 26 , their modes of activation are phylogenetically unrelated 6 , 27 . While E. coli EF-P (EF-P Eco ) strictly depends on ( R )-β-lysylation 22 , 28 – 30 and hydroxylation 31 of a conserved lysine, Pseudomonas EF-P (EF-P Ppu ) is rhamnosylated at an arginine by the glycosyltransferase EarP (EarP Ppu ) at the structurally equivalent position 6 , 21 . Despite these apparent distinct post-translational modifications, a combination of efp Ppu and earP Ppu from P. putida KT2440 can compensate for a lack of efp in E. coli (Δ efp ) as long as the endogenous dTDP- l -Rha pathway remains intact (Fig.…”

Section: Resultsmentioning

confidence: 99%

Two RmlC homologs catalyze dTDP-4-keto-6-deoxy-d-glucose epimerization in Pseudomonas putida KT2440

Koller

Lassak

2021

Sci Rep

Self Cite

View full text Add to dashboard Cite

Abstractl-Rhamnose is an important monosaccharide both as nutrient source and as building block in prokaryotic glycoproteins and glycolipids. Generation of those composite molecules requires activated precursors being provided e. g. in form of nucleotide sugars such as dTDP-β-l-rhamnose (dTDP-l-Rha). dTDP-l-Rha is synthesized in a conserved 4-step reaction which is canonically catalyzed by the enzymes RmlABCD. An intact pathway is especially important for the fitness of pseudomonads, as dTDP-l-Rha is essential for the activation of the polyproline specific translation elongation factor EF-P in these bacteria. Within the scope of this study, we investigated the dTDP-l-Rha-biosynthesis route of Pseudomonas putida KT2440 with a focus on the last two steps. Bioinformatic analysis in combination with a screening approach revealed that epimerization of dTDP-4-keto-6-deoxy-d-glucose to dTDP-4-keto-6-deoxy-l-mannose is catalyzed by the two paralogous proteins PP_1782 (RmlC1) and PP_0265 (RmlC2), whereas the reduction to the final product is solely mediated by PP_1784 (RmlD). Thus, we also exclude the distinct RmlD homolog PP_0500 and the genetically linked nucleoside diphosphate-sugar epimerase PP_0501 to be involved in dTDP-l-Rha formation, other than suggested by certain databases. Together our analysis contributes to the molecular understanding how this important nucleotide-sugar is synthesized in pseudomonads.

show abstract

Section: Resultsmentioning

confidence: 99%

Two RmlC homologs catalyze dTDP-4-keto-6-deoxy-d-glucose epimerization in Pseudomonas putida KT2440

Koller

Lassak

2021

Sci Rep

Self Cite

View full text Add to dashboard Cite

show abstract

“…Except for hydroxylation, none of the modification steps can be bypassed without noticeable effects on translational rescue of PP-proteins. Even modification of EF-P with α-lysine instead of ß-lysine decreases its activity 38 . Notably, the absence of modification in EF-P impairs the translation of polyproline-containing proteins 9 .…”

Section: Discussionmentioning

confidence: 99%

Decrypting the functional design of unmodified translation elongation factor P

Tomasiunaite,

Kielkowski,

Krafczyk

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

Stalling of ribosomes during polypeptide synthesis due to consecutive proline motifs is a challenge faced by organisms across all kingdoms. To overcome this, bacteria employ translation elongation factor P (EF-P), while archaea and eukaryotes rely on a/eIF5A. Typically, these elongation factors become active only after undergoing post-translational modifications (PTMs) such as beta-lysinylation, (deoxy-)hypusinylation, rhamnosylation, or 5-aminopentanolyation. An exception to this rule is found in EF-P members of the PGKGP-subfamily, which remain unmodified. However, the mechanism behind the ability of certain bacteria to avoid metabolically and energetically costly PTMs, while retaining active EF-P, remains unclear. In this study, we investigated the design principles governing the full functionality of unmodified EF-Ps in Escherichia coli. We first screened for naturally unmodified EF-Ps that are active in an E. coli reporter strain. We identified EF-P from Rhodomicrobium vannielii capable of rescuing the growth deficiencies and changes in the proteome of an E. coli Delta epmA mutant lacking the gene for the modifying EF-P-(R)-beta-lysine ligase. We then identified specific amino acids in domain I of the unmodified EF-P variant that affected its activity. Ultimately, we transferred these functional properties to other marginally active members of the PGKGP EF-P subfamily, resulting in fully functional unmodified variants in E. coli. These results have implications for the improved heterologous expression of polyproline-containing proteins in E. coli and offer applications in other bacterial hosts. Understanding the mechanisms that underlie the functionality of unmodified EF-P provides insights into cellular adaptations to optimize protein synthesis.

show abstract

“…A number of studies have employed a range of techniques, including ribosome profiling, mass spectrometry, in vitro and in vivo reporter systems, to identify motifs associated with different degrees of ribosome pausing 14,16,[37][38][39][40][41] . The majority of these studies focuses on motifs which contain at least two prolines, as EF-P is well known to support their synthesis during the transpeptidation reaction 16,38 .…”

Section: Introductionmentioning

confidence: 99%

A versatile dual reporter to identify ribosome pausing motifs alleviated by translation elongation factor P

Tomasiunaite,

Brewer,

Burdack

et al. 2024

Preprint

View full text Add to dashboard Cite

Protein synthesis is influenced by the chemical and structural properties of the amino acids incorporated into the polypeptide chain. Motifs with consecutive prolines can slow down translation speed and cause ribosome stalling. Translation elongation factor P (EF-P) facilitates peptide bond formation in these motifs, thereby alleviating stalled ribosomes and restoring regular translational speed. Ribosome pausing at various polyproline motifs has been intensively studied using a range of sophisticated techniques, including ribosome profiling, proteomics, andin vivoscreenings with reporters incorporated into the chromosome. However, the full spectrum of motifs which cause translational pausing inEscherichia colihas not yet been identified. Here we describe a plasmid-based dual reporter for rapid assessment of pausing motifs. This reporter contains two coupled genes encoding mScarlet-I and chloramphenicol acetyltransferase to screen motif libraries based on both bacterial fluorescence and survival. In combination with a diprolyl motif library, we use this reporter to reveal motifs of different pausing strengths in anE. colistrain lackingefp.Subsequently, we use the reporter for a high-throughput screen of four motif libraries, with and without prolines at different positions, sorted by fluorescence-associated cell sorting (FACS) and identify new motifs that influence translational efficiency of the fluorophore. Our study provides anin vivoplatform for rapid screening of amino acid motifs that affect translational efficiencies.

show abstract

Synthetic post‐translational modifications of elongation factor P using the ligase EpmA

Cited by 7 publications

References 64 publications

Two RmlC homologs catalyze dTDP-4-keto-6-deoxy-d-glucose epimerization in Pseudomonas putida KT2440

Two RmlC homologs catalyze dTDP-4-keto-6-deoxy-d-glucose epimerization in Pseudomonas putida KT2440

Decrypting the functional design of unmodified translation elongation factor P

A versatile dual reporter to identify ribosome pausing motifs alleviated by translation elongation factor P

Contact Info

Product

Resources

About