Archives of Biochemistry and Biophysics

The inactivation of the electrogenic function of the transmembrane sodium transporter in oxidative stress conditions has been intrinsically linked with the oxidation of its catalytically essential thiols. However, the spatial proximity of these catalytically relevant thiols is yet to be fully elucidated and thus still open. Herein, the influence of a thiol cross-linking [diamide, DA (0.1-2mM)] and a thiol alkylating [iodoacetamide, IA (0.1-5mM)] agent on the activity of the synaptosomal Na+/K+-ATPase were determined. In addition, the ability of dithiothreitol to either prevent or reverse the inhibition imposed by the thiol modifiers on the enzyme activity was also evaluated. The results showed that the thiol cross-linker inactivates the electrogenic function of the synaptosomal Na+/K+-ATPase when exposed to the thiols located at either the nucleotide or cation-binding sites. Conversely, irrespective of the exposed active sites, the thiol alkylating agents have no overt effect on the activity of the pump. Furthermore, dithiothreitol markedly prevented but did not reverse the inactivation of the electrogenic pump caused by cross-linking of its critical thiols. Interestingly, both the thiol cross-linker and alkylating agents markedly oxidize dithiothreitol in a time and concentration-dependent fashion. Consequently, within the limit of the present data, it appears that the catalytically relevant thiols of the transmembrane electrogenic pump located at the cationic and nucleotide binding sites, are in close proximity sufficient enough to allow for their cross-linking. HighlightsO_LIThe presence of Na+/K+-ATPase catalytically important thiols at the nucleotide and cationic sites of the enzyme define its vulnerability to oxidative assault. C_LIO_LIThe spatial location of these thiols at vicinal positions at these domains favour the formation of disulphide linkages under oxidative conditions C_LIO_LIThe disulphide crosslinking of these thiols culminate in enzyme inactivation C_LIO_LIThe inactivation can be prevented but not reversed by exogenous thiol compound C_LI

Cu/Zn Superoxide Dismutase 1 (SOD1) is a 32-kDa cytosolic dimeric metalloenzyme that neutralizes superoxide anions into harmless oxygen and hydrogen peroxide. Mutations in SOD1 are associated with ALS, a disease causing motor neuron atrophy and subsequent mortality. These mutations exert their harmful effects through a gain of function mechanism, rather than loss of function. Despite extensive research, the specific mechanism causing selective motor neuron death still remains unclear. A defining feature of ALS pathogenesis is protein misfolding and aggregation, evidenced by ubiquitinated protein inclusions containing SOD1 in motor neurons. This work aims to identify compounds countering SOD1(A4V) misfolding and aggregation, potentially aiding ALS treatment. The approach employed is drug repurposing and in vitro screening of a 1280 pharmacologically active compounds library, LOPAC(R). Using Differential Scanning Fluorimetry Technique (DSF), compounds were tested for their impact on SOD1(A4V) thermal stability. Screening revealed one compound raising protein-ligand Tm by 7{degrees}C, eight inducing a higher second Tm, suggesting stabilzation effect, and five reducing Tm up to 18{degrees}C, suggesting possible interactions or non-specific binding.

The transmembrane protein responsible for the electrogenic transport of Na+ and K+ across the plasma membrane, the Na+/K+-ATPase, highly vulnerable redox modulations and thiol modifying agents due to the presence of thiol groups at the nucleotide and cationic sites. However, reports have demonstrated a preferential interaction of these protein thiols with oxidizing agents. The reactivity of protein thiols is strongly linked with the nature of the microenvironment of these thiols, hence, the present study sought to experimentally elucidate key features of the microenvironment of the catalytically relevant thiols at the substrate-binding sites of this crucial enzyme. Two thiol modifiers with similar thiol-reactive mechanism, but different molecular properties, iodoacetamide (IA) and N-acetyl-4-phenyliodoacetamide (APIAM), were employed. It was observed that while both compounds demonstrated excellent thiol-oxidizing properties in the chemical model, only APIAM had an inhibitory effect on the activity of the Na+/K+-ATPase. The involvement of the catalytically relevant thiols at the nucleotide and cation-binding sites of the enzyme in APIAM-mediated inhibition was confirmed by the protective effect of preincubating the reaction system with dithiothreitol (DTT). The findings from this study suggest that the catalytically relevant thiols of this enzyme are likely buried in a hydrophobic microenvironment. This could be a part of the protective measure of nature for these vulnerable protein thiols. Further details from our findings can be explored in the therapeutic management of diseases for which a dysfunction in the Na+/K+-ATPase have been identified. HighlightsO_LIThe transmembrane Na+/K+-ATPase has well-defined substrate-binding domains exposed to both aqueous microenvironment and buried within the hydrophobic transmembrane microenvironment C_LIO_LIThese microenvironments influence vulnerability of the critical thiols of the enzyme to oxidative assault C_LIO_LIThese thiols are likely buried in the hydrophobic core of the enzyme, thus selecting its susceptibility to thiol modfying agents C_LI

The importance of human aldehyde oxidase (hAOX1) has increased over the last decades due to its involvement in drug metabolism. Inhibition studies concerning hAOX1 are extensive and a common reducing agent, dithiothreitol (DTT), was recently found to inactivate the enzyme. However, in previous crystallographic studies of hAOX1, DTT was found to be essential for crystallization. To surpass this concern another reducing agent used in crystallization trials. Using tris(2-carboxyethyl)phosphine (TCEP), a sulphur-free reducing agent, it was possible to obtain well-ordered crystals from hAOX1 wild type and variant, hAOX1_6A, which diffracted beyond 2.3 [A]. Instead of the typical star-shaped crystals of hAOX1, at pH 4.7, plates are obtained in the orthorhombic space group (P22121) with two molecules in the asymmetric unit. Activity assays with the enzyme incubated with both reducing agents show that contrary to DTT, TCEP does not lead to irreversible inactivation of the enzyme. The replacement of DTT with TCEP in crystallization of hAOX1 provides a strategy to circumvent enzyme inactivation during crystallographic studies, allowing future applications of new assays, such as time-resolved crystallography.

Artemisinin is an effective antimalarial drug sourced from Artemisia annua, but its low and variable yields require enhancement either semi-synthetically or in-planta to meet the global demand for treatment. Though essential enzymes have been identified in the artemisinin biosynthetic pathway, including an essential Cytochrome P450 monooxygenase (CYP71AV1), there are still many unknowns. Cytochrome P450 reductase 1 (herein, AaCPR1), has been experimentally confirmed as an electron transfer partner for CYP71AV1 in its three step oxygenation of key artemisinin precursors. However, the recent discovery of a highly related CPR, herein AaCPR2, introduces the possibility that another, potentially more catalytically favourable interaction, could exist for CYP71AV1. Therefore, enzyme kinetics and differential scanning fluorimetry (DSF) were used in the characterisation of both AaCPR1 and AaCPR2 to determine the existence and source of their catalytic differences. Tested enzyme activity under cytochrome c and NADPH concentrations revealed that AaCPR1 had lower Km and higher kcat/Km values, while AaCPR2 had higher Vmax and kcat values. This suggests that AaCPR1 is more effective at reducing cytochrome c when substrate conditions are limiting, whereas AaCPR2 is more effective than AaCPR1 at reducing cytochrome c when substrate conditions are saturating. This implies a functional partitioning of the two enzymes on the basis of substrate availability. The DSF results provided deeper insight into the different protein-ligand interactions between the two enzymes. AaCPR2 reached lower maximum melting temperatures across all tested conditions, whereas AaCPR1 had higher maximum melting temperatures. Thus, AaCPR1 exhibits higher thermal stability and has a higher temperature threshold than AaCPR2. This contributes to the notion that the AaCPRs are functionally divergent also on the basis of temperature. The cumulative differences in melting behaviour between the two enzymes led to the hypothesis that AaCPR1 and AaCPR2 exhibit different domain motions that may lead to preferential catalysis for one redox partner over another. This was further supported by the prediction of a highly variable loop region between the two enzymes at the connecting domain just after the flexible hinge. If such loops are highly mobile, as predicted, then the residue differences therein could provide a bio-structural basis for the kinetic and thermal/biophysical differences observed between AaCPR1 and AaCPR2. These data support that AaCPR1 and AaCPR2 possess fundamental biophysical differences despite their high degree of relatedness. Ultimately, these differences suggest differential metabolic functions of the two enzyme in artemisinin biosynthesis and/or other important secondary metabolic processes.

Pyranose oxidase from Phanerochaete chrysosporium (PcPOx) is a flavoprotein that forms a covalent 8-N3-histidyl-FAD linkage at His158. Here, we determined the crystal structure of the PcPOx H158Y variant at 1.69 [A] resolution and investigated its FAD occupancy and catalytic activity. Tyr158 adopts a rotamer conformation incompatible with covalent flavinylation, placing its phenolic O atom 8.7 [A] away from the FAD C8 atom. This conformation is stabilized through a Tyr158-Lys79 hydrogen bond. The additional mutation K79A abolishes this hydrogen bond, potentially freeing the Tyr158 residue for other conformations, but does not restore covalent flavin attachment. The H158Y variant retains substantial FAD occupancy and tetrameric assembly but shows markedly reduced oxidase and dehydrogenase turnover rates (less than 13% of WT). These results provide a structural explanation for the failure of tyrosine substitution to support covalent flavinylation in PcPOx and offer insights into requirements for engineering alternative covalent flavin linkages.

In enzymology, hysteresis is manifested as a time-dependent shift in the kinetic behavior of an enzyme. Through hysteresis, the activation or inhibition of a biological pathway can be regulated by a molecule or metabolite that acts as a hysteretic modulator of the enzyme within that metabolic route. This mechanism of regulation contrasts with those that act on gene expression leading to modulation of enzyme protein levels. Through hysteresis, the amplitude of natural oscillations in metabolic pathways can be adjusted according to the levels of a metabolite that might be beneficial for cells. At physiological level, the slow response of hysteretic enzymes to changes, in the cellular levels of substrates, allows a time-dependent buffering effect on certain metabolites. Understanding the mechanisms and properties of hysteretic enzymes has been important for developing new therapies and improving our understanding of these enzymes in biological systems. However, due to their complex kinetics, the study of hysteretic enzymes has remained a challenge over time. In this study, we characterized the reduction of cytochrome b5 by NADH-dependent microsomal enzymes from rat liver using recombinant purified cytochrome b5, coenzyme Q10 and coenzyme Q0, as substrates, to mimic the conditions found in biological membranes, where competition between cytochrome b5 and other substrates might influence their reduction. We found a lag-time-dependent behavior in the cytochrome b5 reduction compatible with the existence of hysteretic modulation induced by endogenous molecules present in these membranes. Our data suggest that at least for the case of coenzyme Q10, fluctuations in its levels may impact metabolic pathways in which reduced cytochrome b5 levels play a key for the function of the cytochrome b5-dependent route.

Risperidone is a second-generation antipsychotic widely prescribed for a variety of psychiatric disorders. Despite its widespread use, its subacute effects on oxidative metabolism in brain and liver tissues remain poorly understood. This study aimed to evaluate the impact of risperidone on antioxidant enzyme activity and lipid peroxidation in rats. A total of fifteen male Holtzman albino rats were randomly assigned to a Control group (n=5, no risperidone) and two treatment groups (n=5 per group) receiving 0.4 mg kg-1 day-1 and 4.0 mg kg-1 day-1 risperidone, administered via orogastric gavage for 20 consecutive days. After treatment, brain and liver tissues were collected. The activity of Superoxide Dismutase (SOD), Catalase (CAT), Glutathione Peroxidase (GPx), Glucose-6-Phosphate Dehydrogenase (G6PDH), and Glutathione S-Transferase (GST) was analyzed. Reduced Glutathione (GSH) levels and lipid peroxidation, measured as thiobarbituric acid reactive substances (TBARS), were quantified. Findings indicate that in brain tissue, both doses significantly increased CAT activity and decreased the SOD/CAT ratio, and that the high dose significantly reduced TBARS levels. In liver tissue, a significant increase in CAT activity was observed with the high dose. Furthermore, both doses significantly increased G6PDH activity and reduced TBARS levels. These results underscore the influence of risperidone on cerebral and hepatic oxidative metabolism during the subacute phase.

Cymoxanil (CYM) is a synthetic acetamide fungicide that has been widely used to combat downy mildew diseases in grapevine cultures and late blight diseases in tomato and potato caused by the oomycetes Plasmopara viticola and Phytophthora infestans, respectively. Despite its extensive application, the biochemical mode of action of CYM remains elusive. Previous reports indicate that CYM affects growth, DNA and RNA synthesis in Phytophthora and inhibits cell growth, biomass production and respiration rate in the well-characterized fungal model Saccharomyces cerevisiae. We therefore used this model to further characterize the effect of CYM on mitochondria. We found that CYM inhibits oxygen consumption in whole cells after 3 h of exposure, which persists over time. Using isolated mitochondria, we demonstrated that CYM specifically inhibits cytochrome c oxidase (CcO) activity during oxidative phosphorylation. Based on molecular docking algorithms, we propose that CYM acts by blocking the interaction of cytochrome c (cyt c) with CcO, hampering electron transfer and inhibiting CcO catalytic activity. Although other targets cannot be excluded, our data offer valuable insights into the mode of action of CYM that can be instrumental to drive informed management of the use of this fungicide.

Enzymes from halophilic organisms have adapted to function in salt concentrations near saturation, making them an interesting model for studying the effect of salt on enzyme catalysis. The main insights into the effect of ionic strength on enzyme catalysis come primarily from enzymes with positively charged surfaces interacting with negatively charged substrates (e.g., ribonucleases), whose activity decreases at high salt concentrations. In this study, we investigated the effect of salt on the kinetics of Glucose-6-phosphate dehydrogenase (G6PDH) from the halophilic archaeon Haloferax volcanii (HvG6PDH), which has optimal activity conditions at concentrations exceeding 2 M KCl. The enzyme catalyzes the NAD+-dependent oxidation of G6P, a negatively charged substrate, and glucose, a non-charged substrate. Using steady-state kinetics, we determined that the enzyme follows an ordered-sequential kinetic mechanism, with NAD+ being the first substrate to bind and NADH being the last released product. Through steady-state kinetic experiments, we found that the main effect of salt is on the KM for G6P, which decreased approximately 50-fold. For glucose dehydrogenase (glcDH) activity, the main effects were a 10-fold increase in kcat and a roughly 10-fold increase in kcat/KM for glucose. To analyze the effect of salt on the different stages of the catalytic cycle, we performed pre-steady-state experiments for both activities. We found that KCl did not affect the catalytic step in G6PDH activity, but it did increase the rate of catalysis in glcDH. Using a minimal model that accounts for substrate binding, chemical transformation, and product release, we determined that the main effect on G6PDH activity was an increase in the rate of G6P association. In contrast, for glcDH activity, the main effect was an increase in the rates of catalysis and product release. The results show that charge screening plays an essential role in the effect of salt on catalysis. Furthermore, it suggests differences in ion penetration to the active site between the two activities. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=190 SRC="FIGDIR/small/632876v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@782511org.highwire.dtl.DTLVardef@1c9bce1org.highwire.dtl.DTLVardef@18f63faorg.highwire.dtl.DTLVardef@ecd1a9_HPS_FORMAT_FIGEXP M_FIG C_FIG

The SLC39A (ZIP) family of zinc ion transporters play a pivotal role in maintaining zinc homeostasis, which is essential for numerous physiological processes involving enzyme catalysis, protein structure and regulation in signal transduction. This investigation employed AlphaFold3 to predict and analyze the 3D structures of all 14 human ZIP family members and revealed key structural and functional features, including transmembrane domains with eight alpha-helices, extracellular and cytoplasmic domains, dimerization, and zinc ion transport pathways. Unique zinc-binding motifs--composed of histidine, aspartic acid, and glutamic acid--were identified. They facilitate zinc ion attraction, selection, and transport. The findings highlight significant structural diversity in these proteins, with additional alpha-helices, disulfide bonds, and other conserved motifs that together contribute to functional specialization across the ZIP family members. Compared to predictions, which exist only for ZIP4, the models incorporate dimeric structures, rationalize loop conformations, and achieve a higher resolution. The predicted 3D structures offer enhanced insights into zinc ion transport mechanisms and provide a foundation for future research into the structural biology of these proteins and their interacting partners in physiology and pathology.

Several rare genetic variations of human XDH have been shown to alter xanthine oxidoreductase (XOR) activity leading to impaired purine catabolism. However, XOR is a multi-functional enzyme that depending upon the environmental conditions also expresses oxidase activity leading to both O {middle dot}- and H O and nitrite ({middle dot}NO -) reductase activity leading to NO. Since these products express important, and often diametrically opposite, biological activity consideration of the impact of XOR mutations in the context of each aspect of the biochemical activity of the enzyme is needed to determine the potential full impact of these variants. Herein, we show that known naturally occurring hXDH mutations do not have a uniform impact upon the biochemical activity of the enzyme in terms of uric acid (UA), reactive oxygen species (ROS) and nitric oxide ({middle dot}NO) formation. We show that the His1221Arg mutant, in the presence of xanthine, increases UA, O2{middle dot}- and NO generation compared to the WT, whilst the Ile703Val increases UA and {middle dot}NO formation, but not O2{middle dot}-. We speculate that this change in the balance of activity of the enzyme is likely to endow those carrying these mutations with a harmful or protective influence over health that may explain the current equipoise underlying the perceived importance of XDH mutations. We also suggest that targeting enzyme activity to enhance the NO2--reductase profile in those carrying such mutations may provide novel therapeutic options, particularly in cardiovascular disease. HighlightsO_LIMutations of xanthine oxidoreductase modulate both its expression and activity C_LIO_LIThe His1221Arg natural mutation increases xanthine oxidoreductase activity C_LIO_LIRaised xanthine oxidoreductase activity coupled with increased availability of nitrite substrate leads to increased NO provision C_LI

Indolamine 2,3-dioxygenase (IDO1) is a hemoprotein that catalyzes the oxidative cleavage of L-tryptophan (L-Trp) to N-formyl-L-kynurenine (L-NFK) along the kynurenine pathway. Its activity depletes L-Trp while initiating a signaling cascade culminating in an immunosuppressive outcome of clinical significance. The generally used in vitro activity assay for IDO1 relies on ascorbic acid and synthetic methylene blue, with the endogenous activator still uncertain. Here we demonstrate sodium hypochlorite, commonly known as bleach, functions as an in vitro activator/cofactor for the recombinant human IDO1-catalyzed dioxygenation reaction. Other hypohalous acids, generated in situ by lactoperoxidase (LPO) or in aqueous solutions of I2 or Br2, also activate IDO1. Contrastingly, the pseudohalide thoicyanate, a known, excellent substrate for LPO, yielded only trace levels of L-NFK. Importantly, complete conversion to L-NFK occurs with sub-stoichiometric hypohalous acid relative to L-Trp, and the overall reaction remains O2-dependent. Kinetic analysis with variable L-Trp and multiple, fixed concentrations of the activators/ cofactors revealed typical Michaelis-Menten kinetics without substrate inhibition, which contrasts past analysis using alternative IDO1 assays. The calculated second order rate constants were overall comparable regardless of the identity and concentration of hypohalous acid. Finally, 1-methyl-L-tryptophan, a reported inhibitor and poor substrate for rhIDO1, was reexamined with the hypohalous acid-dependent conditions revealing an improved catalytic efficiency when compared with the native substrate L-Trp. Along with this unanticipated result, the in vivo functional and mechanistic implications of the newly discovered hypohalous acid-dependent IDO1 activity are discussed. SignificanceIndoleamine 2,3-dioxygenase-1 (IDO1) is a hemoprotein that catalyzes the conversion of L-tryptophan to N-formyl-L-kynurenine (L-NFK). Past efforts have culminated in the conclusion that IDO1 is a checkpoint modulator of mammalian immunity, both in terms of the immunogenicity and immune tolerance. However, the identity of the endogenous activator of IDO1 is still unsettled. Here we report that sodium hypochlorite, aka bleach, activates ferric rhIDO1 in a catalytic manner, enabling rhIDO1 to efficiently catalyze the production of L-NFK in an O2-dependent reaction. The results suggest rhIDO1 can efficiently operate via a hypochlorous acid-dependent mechanism that is reminiscent of the peroxide-shunt pathway used by other hemoproteins. Furthermore, the results suggest a functional role for endogenously produced hypochlorous acid beyond solely killing foreign pathogens.

Protein structure and function dynamics in molecular evolution are intertwined. Theoretical concepts linking structure, function, and evolution of a protein, while often intuitive, necessitate validation through investigations in real-world systems. Our study empirically explores the implications of multiple panE2 gene copies in an organism, shedding light on the functional roles and evolutionary trajectories of Pseudomonas aeruginosas second copy of Ketopantoate reductase (PaKPR2) and its inactivity against the natural substrate Ketopantoate. Evolutionary changes in functional traits were examined around the active site through crystal structures and biochemical analysis. Primarily, apoKPR2 structures reveal a transformed active site cleft, forming a two-sided pocket, while substrate entry is regulated by a molecular gate. Despite cleft closure, molecular interaction properties and activity analysis of PaKPR2 suggest that it can be a versatile keto-acid reductase. However, detailed structural insights from the ligand-bound binary complex of PaKPR2-NADPH and PaKPR2-Ketoisoleucine reveal that the ligand-binding interactions at the active site are conserved and restricted to the molecules of appropriate shape and size that can be accommodated in the available space. Finally, a ternary complex structure, PaKPR2-NADP+-KIC, was solved to understand its functional evolution in terms of the residue microenvironment at the catalytic site. Collectively, the results give detailed visual experiences of different structural perspectives of the proteins functional evolution.

Amyotrophic lateral sclerosis (ALS)-associated mutations in Cu/Zn superoxide dismutase (SOD1) reduce folding stability, resulting in misfolding, aggregation, and ultimately cellular toxicity. A great deal of effort has focused on preventing the misfolding and aggregation of SOD1 as a potential therapy for ALS, however, the results have been mixed. Here, we utilise a small-molecule polytherapy of CuATSM and ebselen to mimic the metal delivery and disulfide bond promoting activity of SOD1s cellular chaperone, the copper chaperone for SOD1 (CCS). We find that polytherapy using CuATSM and ebselen is highly effective at reducing inclusion formation in a cell model of SOD1 aggregation, reduces mutant SOD1-associated cell death, and promotes effective maturation of SOD1 beyond either compound alone. Our data suggest that a polytherapy of CuATSM and ebselen may be an effective method of treating SOD1-associated ALS.

The human mitochondrial amidoxime reducing component 1 (mARC1) is a molybdenum-dependent enzyme whose protein-coding variants confer protection against common metabolic liver diseases. Whereas the frequent A165T variant acts largely through accelerated cellular degradation, the basis of protection by the rarer M187K variant remains obscure, as it has been reported that there are no differences between the "wild-type" and M187K variant protein in terms of cellular protein levels and localisation. Here, the crystal structure of the human mARC1 M187K variant, crystallised as a T4 lysozyme fusion after iterative micro-seeding, was determined at 1.63 [A] resolution. The variant structure is essentially superimposable with the previously reported "wild-type" and A165T structures, with pairwise root-mean-square deviations of 0.3-0.4 [A], and the pentacoordinated molybdenum cofactor is fully intact. Differential scanning fluorimetry across a broad pH range revealed only a modest, pH-dependent decrease in thermal stability associated with the exchange, most pronounced at alkaline pH. These data suggest that the disease-protective effect of M187K is unlikely to originate from gross structural rearrangement or active-site perturbation. SynopsisThe structure of the disease-relevant human mARC1 M187K variant was determined at 1.63 [A] resolution after iterative micro-seeding. The variant does not display relevant perturbations of the overall protein fold or active site structure, but differential scanning fluorimetry detects a pH-dependent decrease in overall protein stability associated with the M187K amino acid exchange.

The mitochondrial pyruvate carrier (MPC) plays a role in numerous diseases including neurodegeneration, metabolically dependent cancers, and the development of insulin resistance. Several previous studies in genetic mouse models or with existing inhibitors suggest that inhibition of the MPC could be used as a viable therapeutic strategy in these diseases. However, the MPCs structure is unknown, making it difficult to screen for and develop therapeutically viable inhibitors. Currently known MPC inhibitors would make for poor drugs due to their poor pharmacokinetic properties, or in the case of the thiazolidinediones (TZDs), off-target specificity for peroxisome-proliferator activated receptor gamma (PPAR{gamma}) leads to unwanted side effects. In this study, we develop several structural models for the MPC heterodimer complex and investigate the chemical interactions required for the binding of these known inhibitors to MPC and PPAR{gamma}. Based on these models, the MPC most likely takes on outward-facing (OF) and inward-facing (IF) conformations during pyruvate transport, and inhibitors likely plug the carrier to inhibit pyruvate transport. Although some chemical interactions are similar between MPC and PPAR{gamma} binding, there is likely enough difference to reduce PPAR{gamma} specificity for future development of novel, more specific MPC inhibitors.

Unlike other bacterial ClpP systems, mycobacterial ClpP1P2 complex is essential for mycobacterial survival. The functional details of Mycobacterium tuberculosis (Mtb) ClpP1P2 remains largely elusive and selectively targeting ClpP of different species is a big challenge. In this work, cediranib was demonstrated to significantly decrease the activity of MtbClpP1P2. By solving the crystal structure of cediranib-bound MtbClpP1P2, we found that cediranib dysregulates MtbClpP1P2 by interfering with handle domain of the equatorial region of MtbClpP1, indicating that the inter-ring dynamics are crucial for its function. This finding provides direct evidence for the notion that a conformational switch in the equatorial handle domain is essential for ClpP activity. We also present biochemical data to interpret the distinct interaction pattern and inhibitory properties of cediranib toward MtbClpP1P2. These results suggest that the variable handle domain region is responsible for the species-selectivity of cediranib, which suggests the equatorial handle domain as a potential region for screening pathogen-specific ClpP inhibitors.

Malate dehydrogenase (MDH: EC:1.1.1.37) catalyzes a key NAD+-dependent redox reaction integral to cellular metabolism. In humans, the cytosolic (hMDH1) and mitochondrial (hMDH2) isoforms operate in distinct compartments, suggesting potential differences in regulation. Here, we present a comparative analysis of hMDH1 and hMDH2 under physiologically relevant conditions, integrating enzymatic assays, ligand binding studies, small-angle X-ray scattering (SAXS), and molecular modeling. Our findings reveal that hMDH2 activity is inhibited by -ketoglutarate, glutamate, NAD+, ATP, and citrate at concentrations consistent with mitochondrial metabolic states characterized by elevated amino acid catabolism or redox stress. Conversely, hMDH1 exhibits minimal impact by these metabolites, with only modest inhibition observed in the presence of ATP and ADP. SAXS analyses confirm that both isoforms maintain stable dimeric structures upon ligand binding, indicating that regulation is not mediated by global conformational changes. Structural modeling and normal mode analyses identify increased flexibility in hMDH1, particularly within the active site loop, thumb loop, and a partially disordered C-terminal helix. In contrast, hMDH2 displays a more rigid architecture and a more electropositive active site environment, correlating with its heightened sensitivity to anionic metabolites. Fluorescence quenching experiments further support these distinctions, demonstrating stronger binding affinities for nucleotide-based ligands in hMDH2 compared to hMDH1. Collectively, these results suggest that isoform-specific regulation of human MDH arises from differences in local structural dynamics and electrostatics, rather than large-scale structural rearrangements. hMDH2 appears adapted to integrate mitochondrial metabolic signals, modulating malate oxidation in response to cellular conditions, while hMDH1 maintains consistent cytosolic function across diverse metabolic states.

BackgroundCytochrome b5 reductase is a flavoprotein that transfers electrons from NADH to multiple electron acceptors, such as cytochrome b5 or ubiquinone. Hysteresis is a phenomenon characterized by a slow transition between active and inactive catalytic states, leading to a lag phase in enzymatic activity. In this study, the effect of the soluble analogue of ubiquinone named 2,3 dimethoxy-5-methyl-1,4 benzoquinone (CoQ0) on the NADH:Cb5 reductase activity of recombinant human soluble Cb5R, using recombinant human soluble Cb5 as a substrate was evaluated. The aim of this study was to determine whether ubiquinone exerts a hysteretic modulation of this activity based on previous studies supporting that microsomal reduction of cytochrome b5 is controlled by redox hysteresis. ResultsThe NADH:cytochrome b5 reductase activity of Cb5R was characterized at different concentrations of Cb5R, cytochrome b5, and CoQ0 by monitoring the reduction of cytochrome b5. The addition of CoQ0 induced the appearance of a lag phase, whose duration increased with the concentration of CoQ0 and decreased with higher concentrations of cytochrome b5 or Cb5R. Additionally, a concentration-dependent decrease in the maximum rate of reduction and the appearance of positive cooperativity was observed in the presence of CoQ0 which resulted in leading to lower KM values for cytochrome b5. This suggests the formation of a CoQ0:Cb5R complex altering the interaction between the reductase and cytochrome b5 which increases the affinity for cytochrome b5. Cyclic voltammetry data support the formation of CoQ0/protein complex that could be responsible for the hysteretic behavior. ConclusionsThese results support the hypothesis that CoQ0 is a hysteretic modulator and inhibitor of the NADH: cytochrome b5 reductase activity of human Cb5R.