Biology subjects

Broetz-Oesterhelt, H.

Publications and source records attributed to Broetz-Oesterhelt, H..

4 recordsLinked to original sources

bioRxiv2023

Bioinformatics-Guided Discovery of Biaryl-Tailored Lasso Peptides

Lasso peptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) that feature an isopeptide bond and a distinct lariat fold. A growing number of secondary modifications have been described that further decorate lasso peptide scaffolds. Using genome mining, we have discovered a pair of lasso peptide biosynthetic gene clusters (BGCs) that include cytochrome P450 genes. Here, we report the structural characterization of two unique examples of (C-N) biaryl-containing lasso peptides. Nocapeptin A, from Nocardia terpenica, is tailored with Trp-Tyr crosslink while longipepetin A, from Longimycelium tulufanense, features Trp-Trp linkage. Besides the unusual bicyclic frame, longipepetin A receives an S-methylation by a new Met methyltransferase resulting in unprecedented sulfonium-bearing RiPP. Our bioinformatic survey revealed P450(s) and further maturating enzyme(s)-containing lasso BGCs awaiting future characterization.

biochemistry↗

bioRxiv2023

Metabolization of alpha-D-carba-glucosamine in vivo generates antimetabolites of cell wall precursors

-D-Carba-glucosamine (CGlcN) is a carbocyclic analog of -D-glucosamine that inhibits growth of Bacillus subtilis and Staphylococcus aureus. CGlcN is internalized and concomitantly phosphorylated via the phosphotransferase system yielding -D-carba-glucosamine-6-phosphate (CGlcN6P), which interferes with expression of the glutamine-fructose-6-phosphate amidotransferase (GlmS; glucosamine synthase) by activating the glmS riboswitch. Herein, we report that CGlcN6P is efficiently metabolized to carbasugar nucleotides along the peptidoglycan biosynthetic route. Mass spectrometric analysis confirmed the occurrence of carbocyclic peptidoglycan nucleotides UDP-carba-D-N-acetyl-glucosamine (UDP-CGlcNAc) and UDP-carba-D-N-acetylmuramic acid-pentapeptide (UDP-CMurNAc-5P) in the presence of CGlcN and revealed accumulation of these carba-metabolites upon antibiotic treatment interfering with biosynthetic enzyme functions. Thus, carbocyclic carbohydrates and nucleotide analogs are generated by the promiscuous bacterial cell wall biosynthetic enzymes and act as antimetabolites, causing bacterial growth inhibition by interference with cell wall synthesis. Our findings reveal CGlcN not only as putative antibiotic molecule with previously unknown antimetabolite mode of action, but also as tool to study the bacterial cell wall metabolism, e.g., in synergy with other antibiotics.

microbiology↗

bioRxiv2022

Uptake of aminoglycosides through outer membrane porins in Escherichia coli

Aminoglycosides are important clinical antibiotics but their molecular uptake mechanism is still not completely understood. Here we quantify and compare the passive transport of three aminoglycosides (kanamycin, gentamicin, and amikacin) across general or sugar specific porins of Escherichia coli (OmpF, OmpC, LamB and ChiP). Our analysis revealed that permeation of aminoglycosides (Kanamycin/Gentamycin/Amikacin) is about the same through ChiP ({approx}5/3/2 molecules/s), OmpF ({approx}10/15/<1 molecules/s) and OmpC ({approx}11/8/<1 molecules/s). In contrast, LamB of smaller pore diameter has no significant permeation ([≤]1/1/1 molecules/s, all values recalculated for a gradient of 10 {micro}M). Biological assays confirmed the relevance of these translocations for antibiotic potency.

biophysics↗

bioRxiv2022

Antibiotic acyldepsipeptides stimulate the Streptomyces Clp-ATPase/ClpP complex for accelerated proteolysis

Clp proteases consist of a proteolytic, tetradecameric core ClpP and AAA+ Clp-ATPases. Streptomycetes, producers of a plethora of secondary metabolites, encode up to five different ClpP homologs and the composition of their unusually complex Clp protease machinery has remained unsolved. Here, we report on the composition of the house-keeping Clp protease in Streptomyces, consisting of a hetero-tetradecameric core built of ClpP1, ClpP2 and the cognate Clp-ATPases ClpX, ClpC1 or ClpC2, all interacting with ClpP2 only. ADEP antibiotics dysregulate the Clp protease for unregulated proteolysis. We observed that ADEP binds Streptomyces ClpP1, but not ClpP2, thereby not only triggering the degradation of non-native protein substrates but also accelerating Clp-ATPase-dependent proteolysis. The explanation is the concomitant binding of ADEP and Clp-ATPases to opposite sides of the ClpP1P2 barrel, hence revealing a third, so far unknown mechanism of ADEP action, i.e., the accelerated proteolysis of native protein substrates by the Clp protease. SignificanceClp proteases are antibiotic and anti-cancer drug targets. Composed of the proteolytic core ClpP and a regulatory Clp-ATPase, the protease machinery is important for protein homeostasis and regulatory proteolysis. The acyldepsipeptide antibiotic ADEP targets ClpP and has shown promise for treating multi-resistant and persistent bacterial infections. The molecular mechanism of ADEP is multi-layered. Here, we present a new way how ADEP can deregulate the Clp protease system. Clp-ATPases and ADEP bind to opposite sides of Streptomyces ClpP, accelerating the degradation of natural Clp protease substrates. We also demonstrate the composition of the major Streptomyces Clp protease complex, a heteromeric ClpP1P2 core with the Clp-ATPases ClpX, ClpC1 or ClpC2 exclusively bound to ClpP2, and the killing mechanism of ADEP in Streptomyces.

microbiology↗