Biology subjects

Lippens, G.

Publications and source records attributed to Lippens, G..

10 recordsLinked to original sources

bioRxiv2025

Selective labeling supports 5-thiooxazole post-translational modification in bufferins

Multinuclear nonheme iron-dependent oxidases (MNIOs) constitute one of the largest families of enzymes involved in natural product biosynthesis. Distinct MNIO subfamilies utilize molecular oxygen to catalyze a wide variety of complex peptide rearrangements, including {beta}-carbon excision and heterocyclization. Highly homologous MNIOs have been proposed to install either oxazolone-thioamides or 5-thiooxazoles as cysteine post-translational modifications in the closely related bufferin and EGKCG families of peptide chalkophores. These alternative structures prompted discussion of the subtle mechanistic features of MNIO enzymes that might determine reaction outcome. Here, we combine uniform 15N labeling with cysteine-specific carbonyl 13C labeling to unambiguously assign 5-thiooxazoles as the cysteine modifications in bufferins. Together with the recent identification of 5-thiooxazoles in three members of the sister EGKCG family and the re-assignment of the cysteine modification in oxazolin, these findings confirm that closely related MNIOs catalyze identical post-translational modifications.

biochemistry↗

bioRxiv2025

Tiarins, a diverse family of natural Trojan-horse aminoacyl-tRNA synthetase inhibitors discovered by genome mining

Trifolitoxin (TFX) is a ribosomally synthesized and post-translationally modified peptide antibiotic produced by Rhizobium anhuiense T24. Although discovered more than half a century ago, its mechanism of action has remained elusive. Here we demonstrate that TFX inhibits arginyl-tRNA synthetase (ArgRS), an essential translation enzyme. TFX acts as a Trojan-horse antibiotic: it enters cells via the inner membrane oligopeptide transporter YejABEF and then undergoes partial proteolysis by the aminopeptidase PepN to release a "warhead" that binds ArgRS and arrests translation. A systematic analysis of prokaryotic genomes revealed that TFX belongs to a widespread family of modified peptides that we designate the tiarins. In addition to ArgRS inhibitors, the tiarin family includes compounds that specifically target at least seven other aminoacyl-tRNA synthetases. These findings define a previously unrecognized family of natural antibiotics and provide a framework for developing a new class of inhibitors targeting various tRNA synthetases.

molecular biology↗

bioRxiv2025

Exploiting Vitamin B6 Dependency: BVL3572S Inhibits HisC and AlaA to Kill Mycobacterium tuberculosis

Tuberculosis remains the leading cause of death from a single infectious agent worldwide, and the growing prevalence of multi-drug resistant Mycobacterium tuberculosis (Mtb) underscores the urgent need for antibiotics with novel mechanisms of action. Here, we characterize BVL3572S, a hydroxamic acid-containing compound that is bactericidal and potently inhibits the growth of both extracellular and intracellular Mtb. Integrated transcriptomic, genetic, and biochemical analyses identified the pyridoxal phosphate (PLP)-dependent aminotransferases HisC (Rv1600) and AlaA (Rv0337c; formerly AspC) as the primary molecular targets of BVL3572S, thereby simultaneously impacting L-histidine and L-alanine biosynthesis. Spontaneous resistance mutants harbored mutations in hisC or alaA. Target engagement was further supported by overexpression studies: AlaA overexpression increased resistance in the presence of L-His whereas HisC overexpression paradoxically increased susceptibility. X-ray crystallography revealed a covalent adduct between PLP and BVL3572S within the HisC active site. The short occupancy of this adduct suggests a futile cycle that sequesters PLP. Isotopic labeling revealed widespread perturbation of amino acid biosynthesis, consistent with PLP starvation. The stepwise resistance observed upon supplementation with L-His and L-Ala together or with PLP alone suggests inhibition of multiple targets. Genome-scale CRISPRi and Tn-seq analyses additionally indicated disruptions in central metabolism, cell envelope integrity, and redox balance, possibly due to PLP depletion cascades. Consistent with its inhibition of AlaA, BVL3572S displayed strong synergy with D-cycloserine, a second-line antitubercular drug targeting D-alanine synthesis and impacting peptidoglycan synthesis, highlighting the potential of this compound in combination therapy. Collectively, our findings establish BVL3572S as a promising lead compound acting through a previously unexploited, multitarget mechanism that induces broad metabolic stress in Mtb, offering a novel therapeutic strategy against drug-resistant tuberculosis.

microbiology↗

bioRxiv2025

Estimating flux dynamics from metabolite concentration time courses with dynafluxr

MotivationQuantifying metabolic fluxes and their dynamics is crucial for addressing fundamental and applied questions in systems and synthetic biology, health, and biotechnology. Since metabolic fluxes are not directly observable, they must be estimated using model-based approaches. While several methods are available to quantify steady-state fluxes, approaches for quantifying flux dynamics remain limited. ResultsWe present a method to quantify flux dynamics from metabolite concentration time courses. Our approach employs constrained B-splines to solve mass balance equations without requiring prior knowledge of catalytic or regulatory mechanisms, or enzymatic parameters. We provide a comprehensive mathematical framework for dynamic flux calculation and demonstrate that our method enables accurate and robust quantification of flux dynamics for both simulated and experimental datasets. Availability and implementationdynafluxr is implemented in R and available on Windows, Unix, and MacOS platforms. The source code and the documentation are freely distributed under GPL-3 license at https://github.com/MetaSys-LISBP/dynafluxr/. A companion notebook producing figures similar to those of this article is available at https://github.com/MetaSys-LISBP/DynafluxR_notebook.

systems biology↗

bioRxiv2025

Multinuclear non-heme iron dependent oxidative enzymes: Landscape of their substrates, partner proteins and biosynthetic gene clusters

Proteins of the multinuclear non-heme iron-dependent oxidative (MNIO) enzyme superfamily catalyze various modification reactions on the precursors of ribosomally synthesized, post-translationally modified peptides (RiPPs). We recently identified two large families of MNIO-modified RiPPs called bufferins, which enhance bacterial growth under copper stress by chelating the excess metal ions. Here, we explored the diversity of potential MNIO substrates by performing extensive in silico studies. Analyses of MNIO-coding biosynthetic gene clusters (BGCs) identified various groups of putative precursors most of which are characterized by specific Cys-containing motifs, throughout the eubacterial phylogenetic tree. The precursors of most MNIO-modified RiPPs harbor N-terminal Sec-dependent signal peptides, a rare feature among bacterial RiPPs. Some precursors are very long relative to those of typical RiPPs, indicating that MNIO enzymes could modify both peptide and protein substrates. We also identified a distinct family of integral membrane proteins with large predicted extra-cytoplasmic domains mostly found in Actinomycetota, frequently but not systematically associated with MNIOs. Most MNIO BGCs harbor genes coding for DUF2063 domain-containing proteins or structurally related proteins, serving as partners of the enzymes for precursor modification. We uncovered a correlation between the presence or the absence of Sec signal peptides in the precursors and the types of partner proteins of the MNIO enzymes. This study depicts the global landscape of potential MNIO-dependent natural products by unveiling groups of peptides and proteins genetically associated with MNIOs. It reveals a treasure trove of potential new RiPP precursors which likely represent a widespread bacterial strategy to deal with copper stress, and most likely other stresses, in natural environments. Impact statementThe multinuclear non-heme iron-dependent oxidative (MNIO) enzymes belong to an emerging superfamily of modification enzymes that catalyze various oxidation reactions on ribosomally synthesized post-translationally modified peptides (RiPP) precursors. The largest families of MNIO-modified RiPPs, called bufferins, are involved in copper homeostasis. In this work we performed extensive in silico analyses to explore the diversity of RiPP precursors genetically associated with MNIO enzymes and identified major families. Some precursors are much larger than typical RiPP precursors, indicating that MNIO enzymes may also modify proteins. We defined subtypes of MNIO enzymes partner proteins dedicated to specific families of precursors. Our analyses of the biosynthetic gene clusters unveiled functions beyond copper homeostasis, likely for the response to other metal stresses. The global landscape of MNIO-modified RiPP precursors will be a basis for investigations into new RiPP families and will undoubtedly lead to the discovery of new modifications and new functions.

bioinformatics↗

bioRxiv2024

MultiNMRFit: A software to fit 1D and pseudo-2D NMR spectra

Nuclear Magnetic Resonance (NMR) is widely used for quantitative analysis of metabolic systems. Accurate extraction of NMR signal parameters - such as chemical shift, intensity, coupling constants, and linewidth - is essential for obtaining information on the structure, concentration, and isotopic composition of metabolites. We present MultiNMRFit, an open-source software designed for high-throughput analysis of one-dimensional NMR spectra, whether acquired individually or as pseudo-2D experiments. MultiNMRFit extracts signal parameters (e.g. intensity, area, chemical shift, and coupling constants) by fitting the experimental spectra using built-in or user-defined signal models that account for multiplicity, providing high flexibility along with robust and reproducible results. The software is accessible both as a Python library and via a graphical user interface, enabling intuitive use by end-users with or without computational expertise. We demonstrate the robustness and flexibility of MultiNMRFit on 1H, 13C, and 31P NMR datasets collected in metabolomics and isotope labeling studies. Availability and ImplementationMultiNMRFit is implemented in Python 3 and was tested on Unix, Windows, and MacOS platforms. The source code and the documentation are freely distributed under GPL3 license at https://github.com/NMRTeamTBI/MultiNMRFit/. Supplementary dataSupplementary data are available online. ContactCyril Charlier (charlier@insa-toulouse.fr) and Pierre Millard (millard@insa-toulouse.fr)

bioinformatics↗

bioRxiv2024

NMR at operational temperature for resonance assignments of a PET degrading enzyme

PETases are enzymes that can break down the poly-ethylene terephthalate (PET) polymer in its constituent building blocks. This enzymatic recycling process offers a sustainable solution for producing new, high-quality plastics from previously used materials. NMR spectroscopy can help in understanding and ultimately improving these enzymes but is always confronted with the lengthy step of acquisition and interpretation of triple resonance spectra for the spectral assignment. Here, we explore whether this step can be made more efficient by recording the spectra directly at high temperature, which simultaneously corresponds to more realistic working conditions for the enzyme. Taking the inactive variant of LCCICCG as an example, we compare spectral quality at 30{degrees}C and 50{degrees}C, and find that the latter condition greatly improves the Signal-to-Noise (S/N) ratio of the standard triple resonance spectra. Going up to 60{o}C, we show that pulse sequences mainly used for the assignment of intrinsically disordered proteins (IDPs) also become feasible. As a result, we present a methodology enabling exhaustive backbone assignment based on a minimal set of triple resonance spectra acquired and analysed in less than two weeks. The assignment process hence can be completed on a time scale comparable to crystallography, bringing NMR in a favourable position to contribute to bio-structural studies on this family of highly thermostable PETases.

biophysics↗

bioRxiv2024

A widespread family of ribosomal peptide metallophores involved in bacterial adaptation to copper stress

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a structurally diverse group of natural products that bacteria employ in their survival strategies. Herein, we characterized the structure, the biosynthetic pathway and the mode of action of a new RiPP family called bufferins. With thousands of homologous biosynthetic gene clusters throughout the eubacterial phylogenetic tree, bufferins form by far the largest family of RiPPs modified by multinuclear non-heme iron-dependent oxidases (MNIO, DUF692 family). Using Caulobacter vibrioides bufferins as a model, we showed that the conserved Cys residues of their precursors are transformed into 5-thiooxazoles, further expanding the reaction range of MNIO enzymes. This rare modification is installed in conjunction with a partner protein of the DUF2063 family. Bufferin precursors are the first examples of bacterial RiPPs found to feature an N-terminal Sec signal peptide and thus to be exported by the ubiquitous Sec pathway, a new paradigm in the RiPP field. Other original features of bufferins are their large size and protein-like fold, which blurs the line between modified peptides and proteins. We reveal that bufferins are involved in copper homeostasis, and their metal-binding propensity requires the thiooxazole heterocycles. Bufferins enhance bacterial growth under copper stress by sequestering excess metal ions in the periplasm. Our study thus describes a large family of RiPP metallophores and unveils a widespread but overlooked metal homeostasis mechanism in eubacteria likely to be relevant to One-Health strategies. Significance statementCopper is both essential and toxic in excess. Bacteria face copper in their environments, notably in phagocytes, hence they have developed several defense mechanisms. We discovered a widespread strategy of protection from copper, through the biosynthesis of natural products that we call bufferins. Bufferins are ribosomally synthesized post-translationally modified peptides (RiPPs), natural products with key roles in bacterial physiology and ecology. Bufferins enhance bacterial growth under copper stress by complexing with the metal using thiooxazole heterocycles that result from enzymatic modification of cysteine residues. With thousands of homologs throughout the eubacterial phylogenetic tree, bufferins represent a highly prevalent strategy of adaptation to metal stress. They are larger in size than most RiPPs, expanding the concept of RiPPs to modified proteins.

microbiology↗

bioRxiv2021

IsoSolve: an integrative framework to improve isotopic coverage and consolidate isotopic measurements by MS and/or NMR

Stable-isotope labeling experiments are widely used to investigate the topology and functioning of metabolic networks. Label incorporation into metabolites can be quantified using a broad range of mass spectrometry (MS)and nuclear magnetic resonance (NMR)spectroscopy methods, but in general, no single approach can completely cover isotopic space, even for small metabolites. The number of quantifiable isotopic species could be increased, and the coverage of isotopic space improved, by integrating measurements obtained by different methods; however, this approach has remained largely unexplored because no framework able to deal with partial, heterogeneous isotopic measurements has yet been developed. Here, we present a generic computational framework based on symbolic calculus that can integrate any isotopic dataset by connecting measurements to the chemical structure of the molecules. As a test case, we apply this framework to isotopic analyses of amino acids, which are ubiquitous to life, central to many biological questions, and can be analyzed by a broad range of MS and NMR methods. We demonstrate how this integrative framework helps to i) clarify and improve the coverage of isotopic space, ii) evaluate the complementarity and redundancy of different techniques, iii) consolidate isotopic datasets, iv) design experiments, and v) guide future analytical developments. This framework, which can be applied to any labeled element, isotopic tracer, metabolite, and analytical platform, has been implemented in IsoSolve (available at https://github.com/MetaSysLISBP/IsoSolve and https://pypi.org/project/IsoSolve), an open source software that can be readily integrated into data analysis pipelines.

systems biology↗

bioRxiv2018

Interact: Automated analysis of protein-ligand interactions by 1D and 2D NMR

NMR titration experiments contain rich information on the thermodynamic, kinetic and structural aspects of protein-ligand interactions. Automated tools are required to process the large number of signals typically acquired in these experiments and facilitate quantitative interpretations. We present Interact, a Python script accessible within the Bruker BioSpin TopSpin software, which allows automated analysis of both 1D and 2D NMR titration experiments. Interact performs peak picking and annotation of the successive spectra and supports quantitative interpretation of changes in chemical shifts and linewidths induced by the ligand (e.g. to estimate dissociation constants) through different fitting procedures. Interact can be applied to all types of 1D and 2D NMR experiments and all nuclei, hence facilitating routine analysis of existing and forthcoming NMR titration data. Interact was implemented in Python and can be used on Windows, Unix and MacOS platforms. The source code is distributed under OpenSource license at http://github.com/MetaSys-LISBP/Interact.

bioinformatics↗