Biochemical characterization and assay development for adenylosuccinate synthetase Rv0357c from Mycobacterium tuberculosis.

BVL3572S inhibits HisC and AlaA, exploiting vitamin B6 dependency to kill Mycobacterium tuberculosis.

The Dephospho-CoA Kinase of Mycobacterium tuberculosis : Molecular Mechanism and Function

Tuberculosis (TB) continues to represent a significant global health danger, with the bacillus Mycobacterium tuberculosis (Mtb) identified as its causative agent. An essential metabolic helper, Coenzyme A (CoA) engages in multiple core cellular processes that are pivotal for both Mtb survival and its ability to cause disease. The terminal step of CoA biosynthesis is catalyzed by dephospho-CoA kinase CoaE, which phosphorylates dephospho-CoA to generate the active cofactor. However, the molecular mechanisms regulating Mtb CoaE catalytic activity remain poorly understood. Here, AlphaFold 3 was used to predict CoaE model. The Ramachandran plot and ProSA were used to validate the model. Subsequently, we performed molecular docking, followed by site-directed mutagenesis. We found that the S15A, R145A or N175A mutants showed markedly reduced activity, whereas the K14A mutation completely abolished activity. Additionally, deletion of residues 27–101 (Δ27–101) led to a significant loss of function. Our findings offer new insights into the structure and mechanism of Mtb CoaE, a promising target for designing selective antituberculosis agents.

Molecular insights into inhibitor action on the catalytic activity of Mycobacterium tuberculosis cystathionine β-synthase enzyme.

Tuberculosis (TB) remains a major global health threat, with Mycobacterium tuberculosis (Mtb) infecting nearly a quarter of the global population. Drug-resistant TB and HIV-TB co-infections emphasize the need for novel therapeutic approaches targeting essential metabolic pathways. Here, we investigated Mtb cystathionine β-synthase (MtbCBS), a pyridoxal 5'-phosphate (PLP) dependent enzyme critical for sulfur metabolism and redox regulation, owing to its potential as a therapeutic target. Despite growing efforts to develop novel therapeutics, the widely used inhibitor aminooxy acetic acid (AOAA) is a non-specific inhibitor of all PLP-dependent enzymes, and the precise structural and mechanistic basis for its activity and specificity remains poorly understood. We present the high-resolution cryo-EM structure of full-length tetrameric MtbCBS in complex with AOAA, revealing a stable PLP-inhibitor adduct stabilized by two highly conserved active-site residues, T75 and Q147. This integrated approach employs cryo-EM, molecular dynamics (MD) simulations, Density Functional Theory (DFT) calculations, and comparative inhibition studies to reveal the molecular basis and determinants governing PLP-enzyme MtbCBS inhibition by AOAA. Through molecular mimic studies, we identified precise structural and electronic features of the inhibitor candidate that are critical for inhibition efficiency. These findings provide a mechanistic rationale for MtbCBS inhibition, and the unexplored roles of these key residues can be considered in the design of next-generation inhibitors targeting CBS enzymes implicated in infectious diseases, cancer, and neurological disorders.

Surveying a Pseudomonas aeruginosa-derived oxidoreductase activity

Findings suggest that the studied FPMO may play a role in antibiotic resistance in P. aeruginosa by oxidatively inactivating ampicillin by oxidatively inactivating ampicillin.

Development of macrocyclic peptide inhibitors of Mycobacterium tuberculosis MurF via mRNA display with genetic reprogramming.

The development of new antibacterials for Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), remains critical due to the continued global burden of disease and the emergence of multi-drug resistant (MDR-TB) and extensively drug-resistant (XDR-TB) strains. The MurF enzyme, which catalyses the ATP-dependent ligation of d-Ala-d-Ala to a UDP-MurNAc-tripeptide precursor in peptidoglycan biosynthesis, represents a promising therapeutic target. In this work, random non-standard peptide integrated discovery (RaPID) mRNA display with genetic reprogramming of a non-canonical uracil-derived amino acid was applied to Mtb MurF, leading to the identification of several high-affinity thioether-linked cyclic peptide ligands, several of which also inhibited the catalytic activity of the Mtb MurX enzyme.

Synthesis, multi-target enzyme inhibition, and in silico evaluation of phthalimide derivatives with relevance to tuberculosis-associated pathophysiology.

Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), remains a significant global health concern requiring innovative therapeutic approaches. Urease is a bacterial enzyme involved in nitrogen acquisition and phagosomal alkalinization in Mtb, whereas thymidine phosphorylase (TP), xanthine oxidase (XO), and butyrylcholinesterase (BChE) are host enzymes associated with nucleotide metabolism, oxidative stress, and cholinergic/immune regulation, respectively, and were included to explore enzyme-inhibitory profiles with potential relevance to tuberculosis-associated pathophysiology. In this study, a new series of phthalimide derivatives was synthesized and structurally characterized through 1H NMR and 13C NMR, with further optimization performed at the DFT level (B3LYP/6-31G). Enzyme inhibition assays demonstrated inhibitory activity across the four targets. Compound C15 emerged as the most effective multitarget enzyme inhibitor, showing strong activity against urease (IC₅₀ = 2.90 ± 0.11 μM), XO (IC₅₀ = 5.61 ± 0.14 μM), and BChE (IC₅₀ = 9.49 ± 0.18 μM), whereas compound C17 displayed the greatest potency towards TP (IC₅₀ = 3.24 ± 0.28 μM). UV-Visible spectrophotometric analysis was conducted to examine enzyme-ligand interactions, revealing concentration-dependent changes consistent with complex formation. Among the series, C17 exhibited the strongest binding affinity towards TP and urease, whereas C15 showed the strongest affinity towards XO. Structure-activity relationship analysis identified benzothiazole, halogen, nitro, sulphonamide, and carboxylic acid functionalities as important contributors to enzyme-inhibitory activity. Collectively, these findings identify C15 and C17 as promising lead compounds for further optimization as multitarget enzyme inhibitors.