Biology subjects

van der Velden, T. T.

Publications and source records attributed to van der Velden, T. T..

4 recordsLinked to original sources

bioRxiv2026

Discovery of a novel chemotype targeting Mycobacterium tuberculosis cytochrome bd through rapid screening and structural elucidation

Antibiotic resistance in Mycobacterium tuberculosis is a pressing global health challenge demanding new therapeutic strategies. The bacterial respiratory chain comprises promising antibacterial targets, with dual inhibition of the terminal oxidases cytochrome bcc:aa3 and cytochrome bd (cyt bd) showing bactericidal activity. While bcc:aa3 inhibitors such as Q203 have advanced clinically, cyt bd remains underexplored due to difficulties in assigning activity of the purified enzyme and structurally resolving the quinol substrate binding site. Here, we report a rapid in vitro screening platform for cyt bd inhibitors by engineering a minimal respiratory system that couples the activity of cyt bd to that of a type 2 NADH dehydrogenase. This coupled assay enables spectroscopic monitoring of NADH oxidation as a proxy for cyt bd activity, allowing rapid screening of over 10,000 compounds. Screening identified WSL017, a fragment with low micromolar potency against both M. tuberculosis and E. coli cyt bd. Kinetic and structural analyses revealed competitive inhibition at the quinol-binding site, providing the first structural insights into cyt bd inhibition by a non-quinone scaffold. WSL017 displayed growth inhibition of M. tuberculosis H37ra, corroborating oxidase inhibition as a promising therapeutic strategy. This work establishes a pipeline for cyt bd inhibitor discovery and highlights new opportunities for structure-guided drug development against cytochrome bd oxidases.

biochemistry↗

bioRxiv2026

Peptide-Induced Formation of Extracellular Vesicles that are DistinctFrom Endogenous E. coli OMVs, and Provide an Enhanced Platformfor Protein Production and Purification.

Bacterial outer membrane vesicles (OMVs), are nano-sized, spherical structures released by Gram-negative bacteria that play diverse roles in bacterial physiology, including communication, nutrient acquisition, and host interactions. These vesicles bud from the bacterial outer membrane and contain lipopolysaccharides, periplasmic proteins, nucleotides, and other biomolecules. The Vesicle Nucleating peptide (VNp) is a short peptide tag that, when fused to the amino terminus of a protein of interest, promotes the formation of bespoke recombinant extracellular vesicles in Escherichia coli, enabling efficient production and simplified purification of recombinant proteins. Here, we characterise VNp-induced vesicles and compare their composition and organisation with naturally produced E. coli OMVs. While both vesicle types possess a single outer membrane-derived lipid bilayer, recombinant protein is highly enriched within the VNp vesicles compared to endogenous OMVs. VNp-fusions and periplasm-targeted recombinant proteins localize to distinct vesicle populations, with VNp-fusions showing markedly higher intra-vesicular concentrations and vesicular purity, compared to the OMV targeted protein. OmpX co-expression further enriched the VNp-fusion content of vesicles, further enhancing yield. The VNp-vesicle lumen is an oxidizing environment, thus supports formation of inter- and intra-molecular disulfide bonds within encapsulated proteins. Overall, VNp-induced vesicles represent a distinct class of recombinant extracellular vesicles that offer a simple and efficient route for producing and purifying concentrated, correctly folded recombinant proteins, expanding the utility of bacterial vesicle systems for biotechnological applications. Significance StatementBacterial extracellular vesicles are recognized as versatile tools for biotechnology yet engineering bacterial vesicle production in a controlled and efficient manner remains challenging. Here we describe how a short Vesicle Nucleating Peptide (VNp) tag, fused to a protein of interest, that can be used to program Escherichia coli to produce recombinant extracellular vesicles that are compositionally and structurally distinct from natural bacterial outer membrane vesicles (OMVs). VNp-induced vesicles are more homogeneous, and more highly enriched in target fusion proteins, providing a simple and efficient route for protein production and purification. The oxidizing lumen of these vesicles supports disulfide bond formation, and rapid compartmentalisation enables expression of otherwise challenging or toxic proteins. This work characterises a distinct class of recombinant bacterial vesicles and establishes a practical platform for producing correctly folded, concentrated, partially purified proteins in a self-packaged form, expanding the potential applications of bacterial extracellular vesicles in biotechnology and synthetic biology.

bioengineering↗

bioRxiv2025

Visualizing the mechanism of quinol oxidation and inhibition of a bd-type oxidase using cryo-EM

Cytochrome bd is a prokaryotic terminal oxidase recognized as an antibiotic target against various pathogens. Despite its critical role in respiration, failure to capture the mechanism of enzyme catalysis and inhibition prohibits structure guided drug discovery. Here, we present cryo-electron microscopy structures of Escherichia coli cytochrome bd-I in monomeric and dimeric forms, along all stages of quinol turn-over and in an inhibitor-bound state. We identify a dynamic Q-loop lid that undergoes a disorder-to-order transition upon substrate binding to the dimer, completing the active site and enabling catalysis. Structure-guided mutagenesis confirms Tyr243 and Arg298 as essential catalytic residues unique to long Q-loop oxidases, highlighting evolutionary divergence from short Q-loop variants. Inhibition by Aurachin D triggers refolding of the active site, occluding substrate access via a conserved Asp239-mediated mechanism. The structural and mechanistic insights presented here establish a comprehensive framework, opening new ways for drug discovery.

biochemistry↗

bioRxiv2024

Menaquinone-specific oxidation by M. tuberculosis cytochrome bd is redox regulated by the Q-loop disulfide bond

Cytochrome bd from Mycobacterium tuberculosis (Mtbd) is a menaquinol oxidase that has gained considerable interest as an antibiotic target due to its importance in survival under infectious conditions. Mtbd contains a characteristic disulfide bond that has been hypothesized to confer a redox regulatory role during infection by constraining the movement of the menaquinone-binding Q-loop. Interference of reductants used in the standard activity assay of quinol oxidases has prevented testing of this hypothesis. Here, the role of the disulfide bond and quinone specificity of Mtbd has been determined by the reconstitution of a minimal respiratory chain consisting of a NADH dehydrogenase and Mtbd, both in detergent and native-like lipid environments. Comparison to cytochrome bd from Escherichia coli (Ecbd) confirms that Mtbd is under tight redox regulation and is selective for menaquinol, unable to oxidize either ubiquinol or demethylmenaquinol. Reduction of the Mtbd disulfide bond resulted in a decrease in oxidase activity up to 90%, depending on menaquinol concentrations. In addition, the catalytic rates of Ecbd and Mtbd are over 10 times lower with the natural lipophilic quinones in comparison to their often-used hydrophilic analogs. Additionally, unlike Ecbd, the activity of Mtbd is substrate inhibited at physiologically relevant menaquinol concentrations. We signify Mtbd as the first redox sensory terminal oxidase and propose that this enables Mtbd to adapt its activity in defence against reactive oxygen species encountered during infection by M. tuberculosis.

biochemistry↗