Biology subjects

Dastmalchi, M.

Publications and source records attributed to Dastmalchi, M..

5 recordsLinked to original sources

bioRxiv2026

Dynamic structural changes and inhibition of insect delta and epsilon glutathione S-transferases by ethacrynic acid and permethrin

Insect glutathione S-transferases (GSTs) play critical roles in xenobiotic detoxification and insecticide resistance, making them promising targets for selective pest-control strategies. Here, we performed a comparative analysis of GSTs representing multiple classes from beneficial insects, agricultural pests, and disease vectors. We found significant isozyme-specific variations in catalytic activity, stability, and conformational dynamics, notably, in relation to inhibition by the commercial chemical agents, ethacrynic acid (ECA) and permethrin (PER). Sequence similarity network analysis revealed distinct clustering of major GST classes and further highlighted the relatively recent evolutionary divergence of the insect-specific delta and epsilon classes. Structural modeling revealed highly conserved glutathione-binding sites (G-site), but substantial variation in the hydrophobic substrate-binding regions (H-site). Further, epsilon-class GSTs exhibited 4 helices oriented approximately 10{degrees} closer to the glutathione-binding site than delta enzymes, suggesting differences in active-site architecture. Steady-state kinetic analyses using 1-chloro-2,4-dinitrobenzene (CDNB) demonstrated that epsilon GSTs are generally more catalytically efficient. Inhibition studies revealed that ECA acts as a potent mixed-type inhibitor of delta-class GSTs, reducing catalytic efficiency by up to 56-fold, whereas PER produced weaker and more species-dependent effects. Notably, ECA binding strongly stabilized delta GSTs, as measured by differential scanning fluorimetry (DSF) and induced a selective rigidification of the 3/4 linker region and active-site motifs, as observed by hydrogen-deuterium exchange mass spectrometry (HDX-MS). Collectively, these findings demonstrate the importance of analyzing the conformational dynamics mediating insect GST inhibition and provide a framework for exploiting isozyme-specific structural features in the design of next-generation selective insecticides.

biochemistry↗

bioRxiv2025

Discovery of pseudobaptigenin synthase, completing the (-)-maackiain biosynthetic pathway

Pterocarpans are structurally complex defence compounds produced by legumes (Fabaceae). They are commonly associated with antimicrobial activity and thought to be synthesized de novo or accumulated in response to microbial pathogens. (-)-Maackiain is a lineage-specific pterocarpan detected in some legumes, including red clover (Trifolium pratense). The biosynthesis of (-)-maackiain involves a distinctive methylenedioxy bridge formation step, predicted to be catalyzed by a cytochrome P450. Specifically, this elusive P450 catalyzes the conversion of calycosin to pseudobaptigenin. We integrated metabolomic and transcriptomic datasets of red clover roots treated with the fungi, Fusarium oxysporum and Phoma medicaginis, to identify candidate P450 genes. Over 40 molecular features were characterized as (iso)flavonoid structures, including the highly abundant O-methylated isoflavones (formononetin and biochanin A), as well as their derivatives. Long infection with P. medicaginis resulted in significant increases in (-)-maackiain, trifolirhizin and other pterocarpans. Concurrently, fungal infections led to upregulation of core and specialized metabolism-related transcripts, including those encoding phenylpropanoid and (iso)flavonoid biosynthetic enzymes. Using weighted gene co-expression network analysis (WGCNA), variance-stabilized expression patterns, and enzyme-class phylogeny, we were able to curate five candidate cytochrome P450s for pseudobaptigenin synthase (PbS) activity, assayed in engineered yeast (Saccharomyces cerevisiae). One candidate P450 was capable of methylenedioxy bridge formation, converting calycosin to pseudobaptigenin and pratensein to 5-hydroxypseudobaptigenin. Therefore, it was renamed T. pratense pseudobaptigenin synthase (TpPbS/CYP76F319). The discovery of TpPbS facilitates the reconstruction of the complete (-)-maackiain biosynthetic pathway and the production of this pterocarpan chemistry at scale for health and agricultural applications. Significance statementO_LIThe discovery of pseudobaptigenin synthase in red clover (CYP76F319), a P450 that catalyzes the formation of a methylenedioxy bridge to convert calycosin to pseudobaptigenin and pratensein to 5-hydroxypseudobaptigenin. C_LIO_LIThe identification of enriched (iso)flavonoids and associated transcriptomic changes in red clover roots in response to two fungi with distinct infection lifestyles (hemibiotrophy and necrotrophy). C_LI

plant biology↗

bioRxiv2025

Chalcone isomerase-like impedes the lactone shunt and enhances flux partitioning in a bifurcated pathway towards isoflavonoid biosynthesis

The reconstitution of biosynthetic pathways in heterologous hosts is often challenged by the switch to a foreign cellular environment, lacking compatible structural or regulatory features. Auxiliary or non-catalytic proteins can play a critical role in modulating metabolic flux and pathway efficiency. Chalcone isomerase-like (CHIL) is a non-catalytic protein known to serve as a partner to chalcone synthase (CHS) in flavonoid biosynthesis, rectifying its promiscuous activity and preventing by-product formation, such as the aberrant p-coumaroyltriacetic acid lactone (CTAL). Here, we extended the characterization of CHILs to the legume-characteristic isoflavonoid pathway. We assessed four CHIL orthologs from diverse plant lineages: Glycine max (GmCHIL), Oryza sativa (OsCHIL), Selaginella moellendorffii (SmCHIL), and Marchantia polymorpha (MpCHIL). Structural modelling suggested that naringenin (flavanone) entry into the CHIL binding cleft may be sterically hindered compared to catalytic CHIs. Moreover, legume CHIL isoforms possess an additional bulky residue, Tyr48, that is expected to impose further constraints on ligand binding. In vitro, CHS produced up to 60% lactone CTAL instead of its desired output; however, CHIL suppressed this aberrant activity to 10%, concomitantly increasing target compound titers. Combinatorial enzyme and yeast biotransformation assays revealed a critical role for CHIL in conducting flux through chalcone, flavanone, and isoflavone biosynthesis. The inclusion of CHIL in our engineered yeast strains enhanced overall titers and, unexpectedly, promoted carbon flux toward the so-called deoxy-branch (isoliquiritigenin, liquiritigenin, and daidzein) by up to 67%, with a 33% increase in final daidzein titers. By extending CHIL characterization to the isoflavonoid pathway, we have revealed an expanded role for this auxiliary protein and underscored its utility in engineered metabolic contexts. Our findings reiterate the often-overlooked impact of non-catalytic proteins in shaping specialized metabolism. HIGHLIGHTSO_LICombinatorial enzyme assays reveal a species-dependent preference for CHILs from more closely related plant phyla by soybean CHS, which improved chalcone and downstream flavanone output by suppressing aberrant lactone formation. C_LIO_LIYeast co-expressing CHIL with the components of the isoflavonoid metabolon exhibited a 67% increase in flux through a legume-characteristic branch of the pathway, resulting in a 33% increase in titers of the major isoflavone, daidzein. C_LIO_LICHIL proteins can be included to engineer the output of phenylpropanoid-derived intermediates preferentially toward isoflavonoid biosynthesis. C_LIO_LIAuxiliary components, such as CHIL, can be designed to refine metabolic composition in bifurcated pathways, as well as enhancing general flux through pathways. C_LI

synthetic biology↗

bioRxiv2024

Serum from patients with Idiopathic inflammatory myopathy induces skeletal muscle weakness

Idiopathic inflammatory myopathies (IIM) are a group of systemic autoimmune inflammatory disorders that primarily affect striated muscles leading to weakness and accelerated fatigue. The disseminated muscle phenotype points to systemic humoral factors as mediators of the disease. Autoantibodies are important biomarkers for disease classification; it is not known if they play a direct role in IIM disease development. This study aims to investigate if IIM patient serum or isolated IgG could directly impair contractile function in muscle. Isolated flexor digitorum brevis (FDB) muscles from healthy mice were exposed to serum (10-50%) from healthy controls or patients with recent onset IIM. Some muscles were exposed to isolated total IgG (50 or 150g/ml) from patients with IIM. Muscle force in whole muscles was measured before and after exposure to sera or IgG. Muscle force and intracellular [Ca2+] in single muscle fibers were measured after exposure to serum. FDB muscles exposed to serum from patients with IIM displayed a marked reduction in force production in both 10% and 50% serum. Moreover, single myofibers dissected from FDB muscles exposed to IIM sera displayed lower force but unaffected Ca2+ release during contractions, which indicates myofibrillar dysfunction, but not intracellular Ca2+ release, as the cause to weakness. FDB muscles exposed to total IgG from IIM patients did not display any reduction in muscle force. In conclusion IIM patient serum, but not total IgG, impairs force production in skeletal muscle fibers. As the experiments were performed in isolated muscle, our results cannot be explained by infiltrating immune cells, impaired neuronal or vascular functions. This suggests that humoral factors play a direct role in the pathogenesis of muscle weakness in recent onset IIM.

physiology↗

bioRxiv2023

In vivo characterization of a secologanin transporter from Catharanthus roseus

Monoterpenoid indole alkaloid (MIA) biosynthesis in Catharanthus roseus is a paragon of the spatiotemporal complexity achievable by plant specialized metabolism. Spanning a range of tissues, four cell types, and five cellular organelles, MIA metabolism is intricately regulated and organized. This high degree of metabolic differentiation requires inter-cellular and organellar transport, which remains understudied. Here, we have fully characterized a vacuolar importer of secologanin belonging to the multidrug and toxic compound extrusion (MATE) family, named CrMATE1/SLTr. Phylogenetic analyses of MATEs suggested a role in alkaloid transport for CrMATE1, and in planta silencing in two varieties of C. roseus resulted in a shift in the secoiridoid and MIA profiles. Subcellular localization of CrMATE1 confirmed tonoplast localization. A full panel of in vivo biochemical characterization using the Xenopus laevis oocyte expression system was used to determine substrate range, directionality, and rate. We can confirm that CrMATE1 is a vacuolar importer of secologanin, rapidly transporting 1 mM of secologanin within 25 min. Notably, the absence of CrMATE1 leads to a transport bottleneck, resulting in the conversion of secologanin to its reduced form, secologanol, both in planta and in the X. laevis system. The unique substrate-specific activity of CrMATE1 showcases the utility of transporters as gatekeepers of metabolic flux, mediating the balance between anti-herbivory potency and cell homeostasis in planta. MIA and secoiridoid transporters could also be deployed in heterologous systems to guide biosynthetic pathways and improve titers of valuable and life-saving MIAs. SIGNIFICANCEWe have fully characterized CrMATE1, a multidrug and toxic compound extrusion (MATE) family transporter in Catharanthus roseus, as a vacuolar importer of secologanin. The translocation of secologanin into the vacuole is necessary for the first committed step of monoterpenoid indole alkaloid (MIA) biosynthesis.

plant biology↗