Jul 2026· Protein Science· Vol 35· 0 citations· 77 references
Medicine
TL;DR
The findings establish M. hassiacum as a valuable model for structural enzymology in mycobacteria and highlight GpgS as a potential drug target, opening new avenues for the development of inhibitors targeting this key, potentially pleiotropic enzyme.
Abstract
Mycobacteria have long posed a threat to human health, being responsible for a range of hard‐to‐treat infections such as tuberculosis, leprosy, and other chronic diseases caused by nontuberculous mycobacteria. Their intrinsic resistance to antibiotics and resilience when exposed to environmental stress complicate treatment and underscore the need for discovering novel drug targets and developing new therapeutic strategies. Mycobacterium hassiacum, a thermophilic mycobacterium, is a promising source of stable proteins suitable for detailed structural analysis. In this study, we investigated its glucosyl‐3‐phosphoglycerate synthase (GpgS), an enzyme that plays a key role in the biosynthesis of methylglucose lipopolysaccharides in Mycobacterium tuberculosis and is implicated in responses to nitrogen starvation and thermal stress in different mycobacterial species. We confirmed the potential of M. hassiacum as a source of enzymes amenable to structural studies, as its GpgS ortholog yielded not only the first mycobacterial GpgS crystallographic structure at near‐atomic resolution (1.10 Å) but also multiple high‐resolution structures under different pH conditions and in complex with various substrates. These structural insights revealed a previously unrecognized aromatic binding pocket that is not only cryptic in nature but also capable of accommodating the substrates p‐aminobenzoic acid and p‐hydroxybenzaldehyde, which have been found to be relevant in its genetic context, thereby establishing a direct link between GpgS activity and broader metabolic pathways in mycobacteria. Our findings establish M. hassiacum as a valuable model for structural enzymology in mycobacteria and highlight GpgS as a potential drug target. The detailed structural data provided here offer a foundation for rational drug design, opening new avenues for the development of inhibitors targeting this key, potentially pleiotropic enzyme.
PA1216, a putative methyltransferase embedded within an NRPS BGC in Pseudomonas aeruginosa strain PAO1 is characterized and a platform for characterizing cryptic gene clusters within secondary metabolic pathways is established for characterizing cryptic gene clusters within secondary metabolic pathways.
Amaan Fruitwala, Jade X Tiszler, Daniel Watson et al.· Protein Science· 0 citations
The Hom family and canilysin are defined as helicolysins, a previously uncharacterized metzincin subfamily distinguished by a conserved Thr-turn and an accessory ND, and implicates these proteins in host-pathogen interactions, adhesion, and immunomodulation.
A. Rodríguez-Banqueri, T. Goulas, Marina Girbal-González et al.· Journal of Molecular Biology· 0 citations
Thiopeptides are ribosomally synthesized and post-translationally modified peptides (RiPPs) that form complex bioactive scaffolds through extensive enzymatic tailoring. The polyglycosylated thiopeptides persiathiacins, exhibit potent activity against multidrug-resistant Mycobacterium tuberculosis (Mtb) and methicillin-resistant Staphylococcus aureus (MRSA). The persiathiacin biosynthetic gene cluster encodes six cytochrome P450 (CYP) enzymes, but the logic of their oxidative modifications was unknown. Here, we establish a protoplast-based genetic system for Actinokineospora and systematically assign functions to all P450s. We demonstrate that PerX hydroxylates the central thiazole, PerV installs the third indole–core crosslink required for macrocyclization, and PerT, not PerU, catalyses indole N-hydroxylation. Combined gene inactivation and metabolite profiling reveal a hierarchical enzymatic sequence leading to the mature scaffold prior to sugar installation. Notably, the intermediate accumulating in the ΩperX mutant exhibits enhanced anti-M. tuberculosis potency compared to persiathiacin A (IC50 = 0.07 vs 1.5 µg mL−1). These results define the enzymatic logic and temporal organization of persiathiacin biosynthesis, providing a conceptual framework for rational diversification of complex thiopeptide natural products.
F. A. Sumang, Maxwell T. Stevens, W. Britton et al.· bioRxiv· 0 citations
ABSTRACT The clinical management of invasive aspergillosis (IA) is limited by the scarcity of antifungal agents and the emergence and spread of drug resistance, which impairs therapeutic efficacy and poses a significant public health threat. Olorofim, a first-in-class clinical antifungal targeting dihydroorotate dehydrogenase (DHODH), represents a promising therapeutic option for IA. It shows activity against several filamentous fungi, including Aspergillus spp., both in vitro and in vivo. Olorofim-resistant Aspergillus fumigatus isolates, characterized by various amino acid substitutions (with a hotspot at G119), have been generated under laboratory conditions through olorofim exposure. However, the potential olorofim resistance mechanisms in Aspergillus flavus, the second-most common pathogen causing IA globally, remain entirely unexplored. In this study, we revealed that the residue G118 in A. flavus, conserved in many filamentous fungi, is homologous to G119 in A. fumigatus by DHODH amino acid sequence alignment. Using a CRISPR-based point mutation approach, we further confirmed that substitutions at the DHODH G118 residue confer acquired olorofim resistance in A. flavus. Additionally, we established a Galleria mellonella infection model and observed that these resistant isolates exhibited no obvious fitness cost, and olorofim treatment was ineffective against infections caused by DHODH G118 mutants. Molecular docking models further indicated that substitutions at the G118 locus may introduce steric hindrance, reducing the binding affinity between olorofim and the DHODH active site and thereby driving resistance. IMPORTANCE Invasive aspergillosis, a life-threatening infection in immunocompromised patients, is increasingly difficult to treat due to limited antifungal options and rising resistance. Olorofim, a novel antifungal targeting dihydroorotate dehydrogenase (DHODH), represents a promising therapy. While resistance in Aspergillus fumigatus has been linked to DHODH G119 mutations, the mechanisms in the globally prevalent pathogen Aspergillus flavus remained unknown. Using CRISPR-based mutagenesis, we demonstrate that amino acid substitutions at G118 confer olorofim resistance, supported by molecular docking showing reduced drug binding. Importantly, olorofim failed to protect larvae infected with resistant mutants in an infection model. These findings provide essential insights for proactively monitoring and managing olorofim resistance in Aspergillus flavus, aiding clinical and public health responses. This study is registered with ClinicalTrials.gov as NCT05101187 and NCT06969703. Invasive aspergillosis, a life-threatening infection in immunocompromised patients, is increasingly difficult to treat due to limited antifungal options and rising resistance. Olorofim, a novel antifungal targeting dihydroorotate dehydrogenase (DHODH), represents a promising therapy. While resistance in Aspergillus fumigatus has been linked to DHODH G119 mutations, the mechanisms in the globally prevalent pathogen Aspergillus flavus remained unknown. Using CRISPR-based mutagenesis, we demonstrate that amino acid substitutions at G118 confer olorofim resistance, supported by molecular docking showing reduced drug binding. Importantly, olorofim failed to protect larvae infected with resistant mutants in an infection model. These findings provide essential insights for proactively monitoring and managing olorofim resistance in Aspergillus flavus, aiding clinical and public health responses. This study is registered with ClinicalTrials.gov as NCT05101187 and NCT06969703.
Hui Xu, Tianyu Liang, Zhongwei Wang et al.· Microbiology spectrum· 0 citations
Aflatoxin B1 (AFB1) is one of the most toxic mycotoxins, widely contaminating agricultural products and posing a serious threat to food safety and human health. Enzymatic degradation is considered a promising detoxification strategy due to its high efficiency, strong specificity, and lack of secondary pollution. AKR13B3, a member of the aldo-keto reductase family, possesses intrinsic catalytic activity for AFB1 degradation; however, its low natural activity severely limits practical application. In this study, the binding mode of the AKR13B3-NADPH complex with AFB1 was first determined using AlphaFold 3.0 and AutoDock Vina. Through interaction analysis, Trp102 and Asp41 were identified as key targets for enhancing catalytic activity. Following site-directed mutagenesis screening, two mutants, D41H and D41T, with significantly improved catalytic activity were obtained, exhibiting 52.32% and 46.44% higher activity than the wild-type enzyme, respectively. Three-dimensional structural simulation revealed that D41H and D41T form stable interactions with the carbonyl group on the lactone ring of AFB1, thereby polarizing the carbonyl group and reducing the activation energy of the reaction, ultimately enhancing catalytic activity. Substrate channel analysis demonstrated that, compared with the wild-type, the D41H and D41T mutants significantly increased the bottleneck radius of the substrate channel (by 25% and 22%, respectively) and shortened the channel length (by 23% and 33%, respectively), thereby partially relieving steric hindrance and diffusion limitations and improving catalytic efficiency. In summary, this study elucidates the molecular basis by which D41H and D41T enhance the catalytic activity of AKR13B3 toward AFB1 through the dual mechanisms of external/hydrogen bond catalysis and channel remodeling, providing an important theoretical foundation for the rational design and directed engineering of AFB1-degrading enzymes.
Qingwei Jiang, Juan Shen, Zhang-Hu Chen et al.· International Journal of Mol...· 0 citations
The rapid spread of antimicrobial resistance, particularly among pathogens such as Staphylococcus aureus and Escherichia coli, highlights the urgent need for novel antibacterial agents with new mechanisms of action. The bacterial enoyl‐acyl carrier protein reductase (FabI), an essential enzyme in fatty acid biosynthesis, represents a promising target for narrow‐spectrum antimicrobials. This study aimed to define consensus pharmacophore models and interaction profiles for FabI through an integrated computational approach. ConPhar and FTMap analyses were applied to experimental structures and molecular dynamics (MD) simulations, while protein–ligand interactions from crystallographic complexes were evaluated using PLIP. Results revealed a conserved binding core involving residues Y156/Y157 and A95 in both species. We also assessed the reliability of residues located in flexible regions by comparing MD snapshots and experimental structures, as well as the contribution of inhibitor–cofactor interactions. Surface mapping identified Y146/Y147 as a key residue, consistent with its reported role in resistance mutations. Additionally, residues I200 (E. coli), V201 (S. aureus), and F203/F204 were identified as potential unexplored interaction sites. Finally, validated consensus pharmacophore models were proposed for future virtual screening and inhibitor design.
P. T. T. F. Leite, Lucas H S Ocarino, G. Veríssimo et al.· ChemMedChem· 0 citations
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