BTZ043, a tuberculosis drug candidate with nanomolar whole-cell activity, targets the DprE1 enzyme of the essential decaprenylphosphoryl-β-D-ribofuranose-2′-epimerase thus blocking biosynthesis of arabinans, vital cell-wall components of mycobacteria. Crystal structures of DprE1, in its native form and in complex with BTZ043, unambiguously reveal formation of a semimercaptal adduct between the drug and an active-site cysteine, as well as contacts to a neighbouring catalytic lysine residue. Kinetic studies confirm BTZ043 as a mechanism-based, covalent inhibitor. This explains the exquisite potency of BTZ043, which, when fluorescently labelled, localizes DprE1 at the poles of growing bacteria. Menaquinone can reoxidize the FAD cofactor in DprE1 and may be the natural electron acceptor for this reaction in the cell. Our structural and kinetic analysis provides both insight into a critical epimerization reaction and a platform for structure-based design of improved inhibitors. Surprisingly, given the colossal tuberculosis burden globally, BTZ043 is the only new drug candidate to have been co-crystallized with its target.
In living organisms, genes encoding proteins that contain flavins as a prosthetic group constitute approximately 2–3% of the total. The fluorescence of flavin cofactors in these proteins is a property that is widely employed for biochemical characterisation. Here, we present a modified Thermofluor® approach called ThermoFAD (Thermofluor®‐adapted flavin ad hoc detection system), which simplifies identification of optimal purification and storage conditions as well as high‐affinity ligands. In this technique, the flavin cofactor is used as an intrinsic probe to monitor protein folding and stability, taking advantage of the different fluorescent properties of flavin‐containing proteins between the folded and denatured state. The main advantage of the method is that it allows a large amount of biochemical data to be obtained using very small amounts of protein sample and standard laboratory equipment. We have explored several cases that demonstrate the reliability and versatility of this technique when applied to globular flavoenzymes, membrane‐anchored flavoproteins, and macromolecular complexes. The information gathered from ThermoFAD analysis can be very valuable for any biochemical and biophysical analysis, including crystallisation. The method is likely to be applicable to other classes of proteins that possess endogenous fluorescent cofactors and prosthetic groups.
form an intersubunit salt bridge. The mutants R292D and D297R are totally inactive. The crystal structure of R292D reveals that the mutant enzyme retains the T-state quaternary structure. However, the mutation induces a reorganization of the interface with the creation of a network of interactions similar to that observed in the crystal structures of R-state yeast and M1 PK proteins. Furthermore, in the R292D structure, two loops that are part of the active site are disordered. The K382Q and R431E mutations were designed to probe the binding site for fructose 1,6-bisphosphate, the allosteric activator. R431E exhibits only slight changes in the regulatory properties. Conversely, K382Q displays a highly altered responsiveness to the activator, suggesting that Lys 382 is involved in both activator binding and allosteric transition mechanism. Taken together, these results support the notion that domain interfaces are critical for the allosteric transition. They couple changes in the tertiary and quaternary structures to alterations in the geometry of the fructose 1,6-bisphosphate and substrate binding sites. These site-directed mutagenesis data are discussed in the light of the molecular basis for the hereditary nonspherocytic hemolytic anemia, which is caused by mutations in human erythrocyte PK gene. ) for its activity. The reaction is essentially irreversible under physiological conditions and is critical for the control of the metabolic flux in the second part of glycolysis. Moreover, the substrate PEP and the product pyruvate are involved in a variety of metabolic pathways. Such a central position in the cellular metabolism is reflected in the regulatory properties of PK, which is a typical allosteric protein (1). The activity is controlled by several physiological effectors, including H ϩ , Mg 2ϩ
SummaryTuberculosis is still a leading cause of death in developing countries, for which there is an urgent need for new pharmacological agents. The synthesis of the novel antimycobacterial drug class of benzothiazinones (BTZs) and the identification of their cellular target as DprE1 (Rv3790), a component of the decaprenylphosphoryl-b-D-ribose 2Ј-epimerase complex, have been reported recently. Here, we describe the identification and characterization of a novel resistance mechanism to BTZ in Mycobacterium smegmatis. The overexpression of the nitroreductase NfnB leads to the inactivation of the drug by reduction of a critical nitro-group to an amino-group. The direct involvement of NfnB in the inactivation of the lead compound BTZ043 was demonstrated by enzymology, microbiological assays and gene knockout experiments. We also report the crystal structure of NfnB in complex with the essential cofactor flavin mononucleotide, and show that a common amino acid stretch between NfnB and DprE1 is likely to be essential for the interaction with BTZ. We performed docking analysis of NfnB-BTZ in order to understand their interaction and the mechanism of nitroreduction. Although Mycobacterium tuberculosis seems to lack nitroreductases able to inactivate these drugs, our findings are valuable for the design of new BTZ molecules, which may be more effective in vivo.
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