NADPH oxidases (NOXs) are the only enzymes exclusively dedicated to reactive oxygen species (ROS) generation. Dysregulation of these polytopic membrane proteins impacts the redox signaling cascades that control cell proliferation and death. We describe the atomic crystal structures of the catalytic flavin adenine dinucleotide (FAD)- and heme-binding domains of NOX5. The two domains form the core subunit that is common to all seven members of the NOX family. The domain structures were then docked in silico to provide a generic model for the NOX family. A linear arrangement of cofactors (NADPH, FAD, and two membrane-embedded heme moieties) injects electrons from the intracellular side across the membrane to a specific oxygen-binding cavity on the extracytoplasmic side. The overall spatial organization of critical interactions is revealed between the intracellular loops on the transmembrane domain and the NADPH-oxidizing dehydrogenase domain. In particular, the C terminus functions as a toggle switch, which affects access of the NADPH substrate to the enzyme. The essence of this mechanistic model is that the regulatory cues conformationally gate NADPH-binding, implicitly providing a handle for activating/deactivating the very first step in the redox chain. Such insight provides a framework to the discovery of much needed drugs that selectively target the distinct members of the NOX family and interfere with ROS signaling.
Benzothiazinones (BTZs) are antituberculosis drug candidates with nanomolar bactericidal activity against tubercle bacilli. Here we demonstrate that BTZs are suicide substrates of the FAD-dependent decaprenylphosphoryl-β-D-ribofuranose 2′-oxidase DprE1, an enzyme involved in cell-wall biogenesis. BTZs are reduced by DprE1 to an electrophile, which then reacts in a nearquantitative manner with an active-site cysteine of DprE1, thus providing a rationale for the extraordinary potency of BTZs. Mutant DprE1 enzymes from BTZ-resistant strains reduce BTZs to inert metabolites while avoiding covalent inactivation. Our results explain the basis for drug sensitivity and resistance to an exceptionally potent class of antituberculosis agents.T he increasing number of drug-resistant Mycobacterium tuberculosis strains that fail to respond to first-and secondline drug treatment demands the development of new antituberculosis drugs. 1−3 Benzothiazinones (BTZs) such as BTZ043 ( Figure 1A) are a promising class of new compounds that kill M. tuberculosis in vitro, ex vivo, and in mouse models of tuberculosis. 4 The minimal inhibitory concentration (MIC) of BTZ043 against M. tuberculosis is 1 ng/mL, which is significantly lower than the MICs of all currently used tuberculosis drugs and drug candidates. Decaprenylphosphoryl-β-D-ribofuranose 2′-epimerase was identified as a BTZ target ( Figure 1B). 4 The enzyme is constituted of DprE1 and DprE2 that together catalyze the epimerization of decaprenylphosphoryl-β-D-ribofuranose (DPR) to decaprenylphosphoryl-β-Darabinofuranose (DPA), the arabinosyl donor for the biosynthesis of mycobacterial cell wall arabinan polymers. 4−6 The reaction is believed to proceed via the keto intermediate decaprenylphosphoryl-D-2′-keto-erythro-pentofuranose (DPX) ( Figure 1B). 5 The Cys387Gly and Cys387Ser point mutations in DprE1 result in 250-and 10 000-fold increases in the MIC, respectively. 4 We previously isolated a covalent adduct of DprE1 and BTZ043 from mycobacteria incubated with BTZ043 and proposed a mechanism of action involving reduction of the essential nitro group of BTZ043 to a nitroso group that then reacts with Cys387 of DprE1 to form a stable semimercaptal ( Figure 1A). 7 However, the mechanism of action of BTZs still poses numerous questions, as it is unclear how BTZs are activated and what the basis of the specificity of the proposed nitroso derivative for DprE1 is. Furthermore, it has not been elucidated whether the observed modification of DprE1 indeed affects the activity of DprE1 or functions by inhibiting the activity of DprE2 (or both); neither have the exact roles of DprE1 and DprE2 in the epimerization reaction been validated.A more detailed characterization of the mechanism of action of BTZs requires the availability of pure DprE1 and DprE2. As our previous attempts to purify recombinant DprE1 and DprE2 of M. tuberculosis H37Rv in their active form were unsuccessful,
Dysregulated ether lipid metabolism is an important hallmark of cancer cells. Previous studies have reported that lowering ether lipid levels by genetic ablation of the ether lipid-generating enzyme alkyl-glycerone phosphate synthase (AGPS) lowers key structural and oncogenic ether lipid levels and alters fatty acid, glycerophospholipid, and eicosanoid metabolism to impair cancer pathogenicity, indicating that AGPS may be a potential therapeutic target for cancer. In this study, we have performed a small-molecule screen to identify candidate AGPS inhibitors. We have identified several lead AGPS inhibitors and have structurally characterized their interactions with the enzyme and show that these inhibitors bind to distinct portions of the active site. We further show that the lead AGPS inhibitor 1a selectively lowers ether lipid levels in several types of human cancer cells and impairs their cellular survival and migration. We provide here the first report of in situ-effective pharmacological tools for inhibiting AGPS, which may provide chemical scaffolds for future AGPS inhibitor development for cancer therapy.
The precursor of the essential ether phospholipids is synthesized by a peroxisomal enzyme that uses a flavin cofactor to catalyze a reaction that does not alter the redox state of the substrates. The enzyme crystal structure reveals a V-shaped active site with a narrow constriction in front of the prosthetic group. Mutations causing inborn ether phospholipid deficiency, a very severe genetic disease, target residues that are part of the catalytic center. Biochemical analysis using substrate and flavin analogs, absorbance spectroscopy, mutagenesis, and mass spectrometry provide compelling evidence supporting an unusual mechanism of covalent catalysis. The flavin functions as a chemical trap that promotes exchange of an acyl with an alkyl group, generating the characteristic ether bond. Structural comparisons show that the covalent versus noncovalent mechanistic distinction in flavoenzyme catalysis and evolution relies on subtle factors rather than on gross modifications of the cofactor environment.
In insects, odorant‐binding proteins (OBPs) connect the peripheral sensory system to receptors of olfactory organs. Medfly Ceratitis capitata CcapObp22 shows 37% identity and close phylogenetic affinities with Drosophila melanogaster OBP69a/pheromone‐binding protein related protein 1. The CcapObp22 gene is transcribed in the antennae and maxillary palps, suggesting an active role in olfaction. Here, we recombinantly produced CcapObp22, obtaining a 13.5 kDa protein capable of binding multiple strongly hydrophobic terpene compounds, including medfly male pheromone components. The highest binding affinity [half maximal effective concentration (EC50) = 0.48 µM] was to (E,E)‐α‐farnesene, one of the most abundant compounds in the male pheromone blend. This odorant was used in cocrystallization experiments, yielding the structure of CcapOBP22. The monomeric structure shows the typical OBP folding, constituted by six α‐helical elements interconnected by three disulphide bridges. A C‐terminal seventh α‐helix constitutes the wall of a deep, L‐shaped hydrophobic cavity. Analysis of the electron density in this cavity suggested trapping of farnesene in the crystal structure, although with partial occupancy. Superposition of the CcapOBP22 structure with related seven‐helical OBPs highlights striking similarity in the organization of the C‐terminal segment of these proteins. Collectively, our molecular and physiological data on medfly CcapOBP22 suggest its involvement in intersex olfactory communication.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.