Archives of Biochemistry and Biophysics

Alkaline ceramidase 2 (ACER2) catalyzes the hydrolysis of ceramides into sphingosine and free fatty acids, playing crucial roles in regulating various biological processes, including but not limited to cell proliferation, survival, and the DNA damage response. However, a pharmacological tool that regulates its catalytic and signaling functions remains to be developed. In this study, N-[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]dodecane-1-sulfonamide (KPB82) was identified as a potent and selective ACER2 inhibitor. In vitro enzymatic assays showed that KPB82 inhibited ACER2 with an IC50 of 190 nM, while displaying minimal activity against acid and neutral ceramidases (IC50> 100 {micro}M) and no significant inhibition of alkaline ceramidase 1 or alkaline ceramidase 3. Enzymatic kinetic studies revealed that KPB82 is a competitive inhibitor of ACER2. The three-dimensional structure of ACER2 was modeled in the AlphaFold3 server, revealing key Zn2+-coordinating histidine residues and a substrate-binding pocket. Molecular docking studies demonstrated that KPB82 binds ACER2 within this pocket, forming hydrogen bonds with the catalytically important amino residues, Asp93 and His215, and interacting with several hydrophobic residues. Lactate dehydrogenase release assays revealed that KPB82 significantly reduced cytotoxicity in HeLa cells overexpressing ACER2, as well as in HCT116 cells, RT4 cells, and human umbilical vein endothelial cells treated with the DNA-damaging agent doxorubicin. Lipidomic analysis showed that KPB82 inhibited sphingosine production in response to ACER2 overexpression or doxorubicin treatment. Collectively, these findings demonstrate that KPB82 is a potent and selective ACER2 inhibitor capable of suppressing ACER2-dependent biological processes and highlight its potential as a tool for studying ceramide metabolism and ACER2-mediated signaling pathways.

Background. Alternative oxidase (AOX) is a mitochondrial terminal oxidase that provides an alternative route for electron transfer, contributing to respiratory flexibility and redox homeostasis under stress conditions. In trypanosomatids, AOX displays a highly uneven distribution associated with extensive diversification of mitochondrial electron transport chains (ETCs). In addition to canonical AOX, many trypanosomatids encode an enigmatic AOX-like protein (AOX-L), whose evolutionary origin and functional significance remain unresolved. Results. Here, we investigated the evolutionary distribution, phylogenetic relationships, structural conservation, and functional divergence of AOX and AOX-L across euglenozoans. Comparative genomic analyses revealed a patchy distribution of both proteins among kinetoplastids, with multiple independent losses during evolution. Phylogenetic analyses demonstrated that AOX and AOX-L represent distinct evolutionary lineages, indicating independent origins rather than duplication-derived divergence. While AOX showed a conserved eukaryotic origin, AOX-L occupied a separate phylogenetic position and lacked key residues required for ubiquinol oxidation. Structural modelling revealed that AOX-L retained the characteristic AOX fold, predicted membrane association, and dimeric organization, but lacked the catalytic architecture necessary for enzymatic activity. Transcriptomic and biochemical analyses of four AOX-containing trypanosomatid species showed that AOX expression and activity correlated with mitochondrial ETC organization. Species lacking cytochrome-dependent complexes III and IV displayed substantially higher AOX expression and enzymatic activity, consistent with AOX functioning as the primary terminal oxidase in these lineages. Conversely, species retaining a canonical ETC exhibited lower AOX activity, suggesting a role in metabolic flexibility and redox regulation. Conclusions. Our study provides a comprehensive evolutionary and functional framework for alternative oxidases in trypanosomatids. We demonstrate that AOX and AOX-L are evolutionarily distinct proteins with different predicted functions: AOX maintains respiratory electron flow according to lineage-specific mitochondrial requirements, whereas AOX-L represents a structurally conserved but catalytically inactive protein family that may have acquired an alternative regulatory role. These findings highlight how mitochondrial respiratory components diversify during eukaryotic evolution and provide a basis for future investigations into the biological function of AOX-L.

Abstract Tricyclic coumarins (TCs) exhibit defensive properties against herbivorous insects and possess valuable pharmacological properties for humans. They are mainly distributed in the Apiaceae, Rutaceae, Moraceae, and Fabaceae families. The chemical structural diversity of TCs in these families primarily arises from differences in skeletal variety and attachment position of the third ring, as observed in unsaturated furanocoumarins (FCs) and saturated dihydrofuranocoumarins (DHFCs). In TC biosynthesis, the FC and DHFC pathways diverge through distinct oxidation patterns. The product specificity of the reported cytochrome P450 monooxygenase at the branching point suggests that these TC groups are produced in nearly identical ratios; however, some species of the genus Angelica (Apiaceae) accumulate DHFCs at higher levels than FCs. In this study, we investigated the enzymatic mechanism responsible for forming these two TC groups using A. keiskei, a Japanese native species used as a medicinal and edible herb. Organ-specific transcriptome analysis highlighted a P450 member CYP71AJ62 that exhibited a close correlation with TC accumulation across organs. Enzymatic characterization demonstrated that CYP71AJ62 predominantly forms a hydroxylated DHFC, vaginidiol, which may promote DHFC over FC skeletal formation in TC biosynthesis. Phylogenetic analysis suggested that CYP71AJ62 has been derived from a gene responsible for FC formation by functional shift without gene duplication. Subsequent three-dimensional structural modeling and site-directed mutagenesis revealed that two amino acid residues on substrate recognition sites cooperatively determine the product specificity of this P450 enzyme. This study provides new insights into the molecular evolutionary process underlying ring-structure diversification and, more broadly, the adaptation of plants to their environments.

Japanese Encephalitis Virus (JEV) belongs to the Flavivirus family, and the RNA dependent RNA polymerase (RdRp) domain located at the C terminus of non structural protein 5 (NS5) regulates de novo viral genome synthesis. The conserved priming loop inserted in the thumb domain of RDRP initiates de novo genome synthesis. Interestingly, the reported replication process initiates with an interaction between NS5 (methyltransferase/RDRP) and NS3 (protease/helicase), and inhibiting this interaction directly correlates with the inhibition of viral replication. The function of the priming loop in the context of the NS5 NS3 interaction is not yet known. In this study, we studied the priming loop function in the NS5 NS3 interaction and viral replication. Using a structure based drug design approach, we screened the Maybridge compound library against the RdRp priming loop and identified 15 candidate compounds based on binding energy. Among them, DSHS00151 showed significant dose dependent inhibition of the NS5 NS3 interaction with an IC50 of 3.5 micromolar in the mammalian two hybrid assay, inhibited viral infectivity with an IC50 of 2.54 micromolar, and reduced viral RNA load with an IC50 of 2.9 micromolar, compared to CD10712. Molecular Dynamics (MD) simulations revealed that DSHS00151 exhibits stronger and more stable binding with the priming loop residues (790 to 812) compared to CD10712. The molecular mechanism suggested by the docking of the NS5 and NS3 proteins showed that the priming loop tends to rotate toward the NS3 protein to stabilize the complex, and compound binding restricts this loop rotation, thus compromising the stability of the NS5 and NS3 complex and affecting viral replication. Overall, our study validated the function of the NS5 priming loop as an allosteric site, and compounds binding to this allosteric site belong to the Non Nucleoside Reverse Transcriptase Inhibitors (NNRTI) class of inhibitors, which disrupt the NS5 and NS3 interaction and could be developed as novel anti JEV therapeutics.

The sulfur mobilization (SUF) system is the only Fe-S cluster biosynthesis pathway in Mycobacterium tuberculosis (M. tb) and Mycolicibacterium smegmatis (M. smegmatis). In this pathway, SufC protein acts as an ATPase in the SufBC2D scaffold for Fe-S cluster biogenesis. This crucial role positions SufC as a promising target for the development of antimycobacterial therapeutics. However, the SufC ATPases from Mycobacterial species are not yet characterized. This study includes cloning, expression, purification, and characterization of SufC homologs from M. tb (Rv1463) and M. smegmatis (MSMEG_3124) using biochemical, biophysical, and computational approaches. Both SufC proteins were purified using affinity and size-exclusion chromatography and observed to exist as monomers in solution. Although both proteins have the conserved domains, they exhibit different ATP-dependent kinetic efficiencies, demonstrating differences in their local environment and catalytic behavior. Furthermore, CRISPR interference targeting MSMEG_3124 results led to diminished M. smegmatis growth, highlighting the critical role of SufC in cellular viability. Modeling and virtual screening led to the identification of three candidate compounds, of which Z2911048515 and Z66741142 were selected for experimental evaluation. Circular dichroism spectroscopy analysis revealed slight differences in the spectral features of Rv1463 in the presence of Z2911048515 and Z66741142, while significant spectral variations were observed for MSMEG_3124. Importantly, Z66741142 was able to cause changes in the fluorescence spectra of both SufC protein homologs. Surprisingly, both compounds showed only limited inhibition of SufC ATPase activity, exhibited distinct modes of action, and may serve as preliminary chemical scaffolds for optimization and testing as effective inhibitors. This study presents functional diversity of mycobacterial SufC proteins across pathogenic and non-pathogenic species and provides a basis for developing a framework for further mechanistic and structure-guided investigation within the indispensable SUF pathway.

Sulfation plays an important role in many metabolic processes such as substrate detoxification, solubilization or activation. The enzymes responsible for these reactions, sulfotransferases, occur in all domains of life and can be classified in two major, evolutionary independent groups, PAPS dependent and PAPS independent or arysulfate sulfotransferases (ASST) enzymes. Besides the fact that they play key roles in biological functions, sulfotransferases can be interesting enzymatic tools to selectively sulfate specific positions of various metabolites. To this end we need to understand the relation between sequence, structure and function. Actually, eukaryotic PAPS dependent sulfotransferases have extensively been studied, while we largely lack knowledge about the diversity of bacterial PAPS-independent ASSTs. We have conducted a multidisciplinary study of a bacterial sulfotransferase from Salmonella typhimurium, revealing the importance of active site residues for activity and guiding substrate specificity. Based on the crystal structure in complex with substrate, we used molecular docking combined with Quantum Mechanics/Molecular Mechanics (QM/MM) free energy calculations to investigate the reaction catalytic mechanism with a focus on the role of specific active site residues in substrate specificity. Our results show that the catalytic mechanism involving three conserved histidines is common with previously determined ASSTs, and highlight a specific pair of aromatic residues, Phe174 and Tyr557, that play an important role in defining the substrate specificity in ASST clade 5. These findings enhance our general understanding of how substrate specificity is attained and has the potential to guide further enzyme engineering to expand the acceptor scope of ASSTs.

[FeFe]-hydrogenases are molecular hydrogen (H2) converting enzymes that employ a hexanuclear iron-complex, the H-cluster, as catalytic cofactor, and a proton transfer pathway (PTP) that allows efficient proton coupled electron transfer (PCET). Recent phylogenetic analyses revealed different groups of [FeFe]-hydrogenases. Although only very few members from other groups have been characterized, current knowledge suggests high catalytic activity is predominantly associated with group A members. Here, we show that metal ions inhibit group A [FeFe]-hydrogenases by binding to three specific residues at the entrance of the PTP. Exchanging residue H565 of Clostridium pasteurianum CpI results in a metal-insensitive protein variant with wildtype like activity. In contrast, exchanging S320 and H569 in CpI, and their counterparts in additional group A [FeFe]-hydrogenases results in enzymes with strongly decreased activities and large overpotential requirements. These features are consistent with important roles of both residues in catalytic proton transfer. CpI structures reveal that H569, locally anchored by E278, can be present in two conformations so that we propose a histidine-dependent pendulum mechanism for exchanging protons between bulk solvent and the PTP, which is well supported by theoretical calculations. By that, our study widens information on how the mobility of histidine, governed by residues in the vicinity, contributes to the fundamental concept of PCET.

Human Ca2+/calmodulin (CaM)-dependent protein kinase II alpha (CaMKII) is a highly abundant and conserved protein in the brain. It is a multifunctional serine/threonine kinase that phosphorylates cellular substrates across subcellular compartments, activating signaling pathways essential for long-term memory storage. However, a detailed understanding of protein structure and enzyme-substrate interaction mechanisms require a method to produce active, stable, and pure proteins at scale. Since phosphorylation can alter catalytic activity and substrate affinity, examining the non-phosphorylated state enables an unbiased characterization of basal enzymatic function. Therefore, producing the non-phosphorylated form of the kinase is important for biochemical and structural studies aimed at understanding kinase activation mechanisms. We describe a detailed protocol for the expression and high-yield purification of full-length, non-phosphorylated human CaMKII in bacterial cells. CaMKII was co-expressed with {lambda}-phosphatase to maintain it in a non-phosphorylated state. Protein expression was induced by autoinduction at low temperature to enhance the stability and solubility of CaMKII. Recombinant CaMKII was purified by Ni-NTA affinity chromatography, followed by gel filtration chromatography. Although CaMKII has been considered difficult to purify in its non-phosphorylated form, we obtained it in high yield, and the protein was predominantly non-phosphorylated. Our results show that combining co-expression with autoinduction improves both the solubility and yield of the homogeneous non-phosphorylated form of CaMKII, which could be suitable for subsequent biochemical and structural studies.

Antifreeze proteins and peptides adhere to ice crystals and inhibit their growth, resulting in freezing point depression. AFP6 is an abundant antifreeze peptide in the blood serum of winter flounder (Pseudopleuronectes americanus). This 37-residue peptide forms an amphiphilic -helix with a hydrophobic ice-binding surface. In this study, prediction of the peptide structure suggested the formation of an antiparallel dimer burying the hydrophobic surfaces of both subunits. The oligomerisation of AFP6 was therefore examined in aqueous solutions using size-exclusion chromatography and chemical crosslinking along with ice-binding evaluation. Crosslinking of non-amidated AFP6, followed by denaturing electrophoresis, revealed bands consistent with monomers and dimers, whereas results for the amidated form and a recombinant mutant suggested additional larger forms. Bands consistent with the presence of monomers and dimers were also predominant following non-amidated AFP6 crosslinking under a variety of solution conditions, with smaller proportions of peptide assembled up to tetrameric sizes; however, the larger oligomers appeared to be absent from AFP6 treated with sodium bicarbonate. Isolated oligomers of non-amidated AFP6 produced ice crystal morphologies and thermal hysteresis comparable to those for the unfractionated peptide suggesting similar interaction with ice. Oligomerisation of AFP6 may allow greater solubility of this amphiphilic -helical peptide in aqueous solutions.

Nitrate is an important oxyanion in bacterial physiology, with major roles as a nitrogen source for assimilation and as an alternative electron acceptor in anaerobic respiration. Accordingly, diverse nitrate-responsive regulatory systems sense nitrate availability and control the expression of genes involved in nitrate transport and metabolism. In the NreABC system, the soluble GAF-like domain protein NreA functions as a cellular nitrate sensor. To date, our molecular understanding of nitrate recognition by NreA has been derived primarily from studies of the Staphylococcus carnosus protein, with less known about homologues from other species. Here, we identified 736 NreA homologues through bioinformatic analysis of the GAF-like domain superfamily and selected five staphylococcal representatives for thermodynamic characterization by isothermal titration calorimetry. Despite conservation of the nitrate-binding pocket, these homologues resolved into high- and low-affinity groups with distinct thermodynamic profiles. Comparative structural modeling and chimeragenesis of a low-affinity homologue revealed that the C-terminal region is a non-coordinating determinant of nitrate-binding affinity. Together, these findings show that a conserved nitrate-binding pocket preserves recognition, while distal sequence variation shapes thermodynamics and tunes the nitrate concentration range over which NreA sensors can function.

Calcium (Ca2+) is a universal second messenger that orchestrates plant growth, development, and stress responses. Plant calcineurin B-like (CBL) proteins sense and relay Ca2+; signals in response to pathogens to activate downstream defense signalling pathways. Early blight disease, caused by the necrotrophic fungal pathogen Alternaria solani, affects tomato crops and severely limits their productivity. In the present study, we found that the tolerant tomato cultivar (Arka Rakshak) restricts A. solani infection and disease progression compared to the susceptible cultivar (PKM-1). Comparative transcriptome and qRT-PCR analysis of A. solani-infected tomato leaves showed that SlCBL1-2 (Solyc08g007160.3), a Ca2+; sensor, is up-regulated in the tolerant cultivar, both relative to its own uninfected control and the infected susceptible cultivar, whereas it was not significantly induced in the uninfected susceptible cultivar. Protein sequence analysis indicates that SlCBL1-2 contains four EF-hand motifs, an N-terminal N-myristoylation/S-acylation module and a C-terminal FPSF motif, and it is predicted to interact with several CBL-interacting serine/threonine protein kinases (CIPKs). However, the evolutionary analysis, conformational transitions upon binding to Ca2+, and the Ca2+-binding affinity of SlCBL1-2 have not been previously characterized. Molecular Dynamics (MD) simulation of the AlphaFold structure of SlCBL1-2 indicates a well-folded structure and binding of two Ca2+ ions at EF-hands 3 and 4. Further, far-UV circular dichroism and tryptophan fluorescence spectroscopy of recombinant SlCBL1-2 indicate a natively folded structure in the absence of Ca2+ and undergo minimal secondary and tertiary structural changes upon Ca2+-binding, in contrast to several Ca2+-binding proteins. Of note, SlCBL1-2 binds Ca2+ with apparent KD values of 526 {+/-} 113 {micro}M and 28.7 {+/-} 9.47 {micro}M, as evidenced by isothermal titration calorimetry. Together, our study provides an experimental description of the Ca2+-binding properties of tomato CBL protein and defines the molecular basis by which pathogen-induced SlCBL1-2 may act as a Ca2+ sensor in defense signalling.

The dopamine D2 receptor is a principal target of antipsychotic and antiparkinsonian drugs, yet well-characterized fluorescent ligands for the D2 receptor remain scarce compared with its relative, the D3 receptor. Here we show that fluorescent ligands developed for the D3 subtype can be repurposed as tools for studying the D2 receptor pharmacology. Using a machine-learning-assisted, live-cell fluorescence microscopy assay, we screened twelve fluorescent probes and identified eight ligands that bound specifically to the D2 receptor at 10 nM. Four ligands exhibited classical saturable binding, yielding Kd in the nanomolar range, whereas the remaining probes displayed unsaturated, time-dependent binding profiles. Kinetic analyses revealed substantial differences in association and dissociation behavior, highlighting the importance of time-resolved measurements. Competition binding with dopamine and haloperidol yielded affinity estimates consistent with literature values. These results expand the toolkit of validated D2 receptor fluorescent probes and support cross-receptor repurposing as a strategy for fluorescent ligand development.

Thermophilic bacterial lipases must combine structural stability with the conformational flexibility required for interfacial activation, yet the local organization of their metal-binding sites remains incompletely understood. Here, we determined two X-ray crystal structures of the family I.5 lipase from Geobacillus kaustophilus (GkLip). The 2.47-[A] orthorhombic model contains a Ca2+ ion at the conserved metal-binding site, whereas the 2.30-[A] anisotropic triclinic model contains four non-equivalent protein chains in which no Ca2+ ion was modeled. GkLip retains the canonical /{beta}-hydrolase fold, the Ser114-Asp318-His359 catalytic triad, the Phe17/Gln115 oxyanion-hole region, an extended lid, and a conserved Zn2+-binding site. In the orthorhombic structure, Gly287, Glu361, Asp366, and Pro367 contribute to the Ca2+ coordination environment. Pro367 adopts a C{gamma}-endo pucker in the Ca2+-containing structure; three Ca2+-free chains also adopt the endo pucker, whereas one adopts a C{gamma}-exo pucker. The presence of both Pro367 pucker states in the Ca2+-free structure is consistent with calcium preferentially stabilizing a pre-existing, coordination-compatible conformation. This local organization is further supported by an Arg35-centered network of direct and water-mediated contacts. Comparison with the 94%-identical open G. thermocatenulatus lipase reveals substantial lid rearrangement despite strong conservation of the protein core. Together, these structures highlight localized conformational variability at the calcium-binding region within an otherwise highly conserved thermophilic lipase scaffold.

Abstract Background: Chronic subdural hematoma (cSDH) remains a significant and increasingly prevalent cause of morbidity in older adults. Disease progression is sustained by de novo formed highly vascularized pseudomembranes and a persistent cycle of fluid accumulation and hematoma expansion. Although inflammatory and angiogenic pathways have been implicated in cSDH pathogenesis, the endothelial mechanisms underlying persistent vascular leak remain poorly understood. We investigated the cSDH vascular microenvironment in human patients and examined the role of vascular endothelial cadherin (VECAD) remodeling in cSDH endothelial dysfunction. Methods: Human cSDH membranes and fluid were evaluated by multiplex profiling, histology, immunofluorescence, immunoprecipitation, western blotting and exploratory bulk RNA sequencing. A human brain endothelial cell (HBEC) line was exposed to patient-derived cSDH fluid to assess VECAD remodeling, endothelial barrier permeability, and proliferation. Results: Multiplex profiling of patient-derived cSDH fluid detected mediators associated with endothelial cell (EC) activation, extracellular matrix remodeling, inflammation, and angiogenesis. In patient pseudomembranes, discontinuous VECAD coincided with erythrocyte extravasation. Two hyperdense (high-risk) specimens showed more extensive disruption than the hypodense (low-risk) specimen while vessel counts appeared similar. Bulk RNA sequencing identified an angiogenic environment and enrichment in cadherin regulation. A soluble VECAD (sVECAD) isoform was detected in cSDH fluid. Exposure of HBECs to patient-derived cSDH fluid increased EC proliferation, cytoplasmic VECAD (cVECAD) accumulation, and EC barrier permeability. Concurrent dexamethasone or minocycline treatment attenuated VECAD cleavage. Conclusions: VECAD disruption is a prominent feature of cSDH and is associated with EC barrier dysfunction. These findings support further evaluation of soluble VECAD as a candidate biomarker and of junction-preserving pathways as potential therapeutic targets.

Purpose. Neuroblastoma is the most common malignancy of infancy, and cancer cells strongly depend on iron metabolism for proliferation. Whether sub-cytotoxic iron load reprograms mitochondrial and redox metabolism, and how this relates to the malignant behavior of neuroblastoma cells, is poorly defined. We hypothesized that exposure of human neuroblastoma cells to a non-toxic iron overload perturbs mitochondrial and redox metabolism, thereby affecting cancer cell properties. Methods. SH-SY5Y neuroblastoma cells were exposed to 100 M iron sulfate for 24 h. The resulting changes were monitored using complementary methods, including cell viability, cytotoxicity, proliferation and migration assays, intracellular iron quantification by ICP-MS, confocal imaging of mitochondrial morphology and membrane potential ({Delta}{Psi}m), high-resolution respirometry with flux control ratios, probe-based oxidant and H2O2 measurements, and label-free quantitative proteomics with Ingenuity Pathway Analysis. Results. Non-toxic iron loading raised intracellular iron about four-fold, promoted cell proliferation and reduced migration. Iron induced mitochondrial fragmentation and {Delta}{Psi}m depolarization while preserving mitochondrial content and OXPHOS coupling efficiency. Among respiratory states, only proton-leak respiration increased, paralleled by a higher leak/ETS coupling ratio. Total cellular oxidant levels and H2O2 output were strongly reduced. The proteome showed downregulation of ribosomal proteins and translation/RNA-processing components and upregulation of histones and ferritin. Conclusion. Mild iron exposure reprograms mitochondrial and redox metabolism in neuroblastoma cells. The observed selective increase in proton leak lowers {Delta}{Psi}m to a range that biophysically limits oxidant production while OXPHOS coupling is preserved. Interfering with this adaptive, proton leak-dependent and preventive antioxidant program may offer a way to alter neuroblastoma progression.

Lipoate is an essential protein-bound cofactor attached to conserved lysine residues in structurally related acceptor proteins, including GcvH of the glycine cleavage system and LbpA proteins required for sulfur oxidation by the sulfur-oxidizing heterodisulfide-like (sHdr) complex. The coexistence of distinct lipoylation pathways and acceptor proteins raises the question of how the enzymes initiating lipoate attachment discriminate between homologous but functionally distinct substrates. We systematically tested lipoate:protein ligases and potential acceptor proteins from five representatives of the bacterial phyla Pseudomonadota and Aquificota using native gel-shift assays and MALDI-TOF mass spectrometry. The canonical Escherichia coli lipoate:protein ligase Lpl(AB) preferentially modified GcvH proteins but showed little or no activity toward LbpAs. In contrast, sulfur oxidation-associated lipoate:protein ligases, termed sLpl(AB), which function in the sLpl(AB)-LipS1/LipS2 lipoate assembly pathway, strongly preferred LbpA proteins and generally failed to modify GcvH. Conserved cysteine residues characteristic of LbpAs were dispensable for recognition by sLpl(AB). Instead, comparison of amino acid sequences and electrostatic surfaces revealed predominantly acidic interaction surfaces in GcvH proteins and corresponding basic regions in LbpAs. Accordingly, replacement of selected basic regions in Roseovarius mucosus LbpA2 by acidic residues reduced modification by its cognate sLpl(AB) and simultaneously enabled weak modification by E. coli Lpl(AB). These findings establish electrostatic surface properties as major determinants of substrate discrimination by lipoate ligases and show that recognition depends on distributed features of the folded acceptor protein rather than solely on the sequence surrounding the lipoylated lysine.

Methylmalonic aciduria (MMA) is caused by deficiency of methylmalonyl-CoA mutase (MMUT), which catalyses the final step of propionate catabolism and fuels the tricarboxylic acid cycle for energy production. Previous studies reported disrupted mitochondrial homeostasis in MMA, including reduced mitochondrial membrane potential and increased oxidative stress, especially in energetically demanding and chronically affected tissues like brain and kidneys. However, how these changes impact mitochondrial energy production remains unclear. Here, we systematically investigated mitochondrial energy production using extracellular flux analysis in cellular models of MMA, including 293T cells, patient-derived fibroblasts, urine-derived epithelial kidney cells and induced pluripotent stem cells (iPSCs) as well as iPSC-derived neurons harbouring either complete loss (knockout, KO) or pathogenic missense variants in MMUT. We found no impact on mitochondrial energy production in MMUT-KO 293T cells and fibroblasts compared to controls. In contrast, fibroblasts and 293T cells expressing the pathogenic MMUT-p.N219Y variant showed decreased energy production. This corresponds to the altered mitochondrial membrane potential in 293T cells, but contrasts with their unchanged mitochondrial abundance. Depletion of glucose, glutamine and pyruvate from the media or provision of each individually as sole fuel source exacerbated the phenotype of MMUT-p.N219Y clones, but did not induce a phenotype in MMUT-KO 293T cells. Finally, we confirmed reduced mitochondrial energy production in patient-derived kidney cells but found no evidence in MMUT-p.N219Y iPSCs and their derived neurons. Overall, our work suggests that the impact of MMUT-deficiency on mitochondrial energy production is cell type- and variant-dependent. Further investigations should clarify molecular mechanisms and their clinical impact.

Exploring the ability of metabolic enzymes to interact with diverse small molecules can provide insights into their ligand recognition and activity regulation. Glucose-6-phosphate isomerase (GPI) is an enzyme that catalyzes the second step of glycolysis, a critical node that links glucose metabolism to other cytosolic pathways, such as pentose phosphate and hexosamine biosynthetic pathways. Despite its importance in cellular metabolism, little is known about the broad ligandability of GPI. Here, we performed a structure-based virtual screening using molecular docking to identify new modulators predicted to engage with the human GPI active site. Enzyme kinetic and binding studies confirmed eleven compounds as active-site binders of GPI, exhibiting inhibitory activities and binding affinities in upper micromolar to sub-millimolar range. The binding of several ligands to GPI active site was further supported by X-ray crystallographic data. In addition, in vitro assays were performed to evaluate the modulatory effects of the GPI hits on cellular glycolysis and viability. The identified inhibitors included the mono-phosphorylated metabolite of ribavirin, which inhibited glycolytic flux and cell viability; two compounds with phosphate bioisosteres; and endogenous phosphate-containing metabolites from the pentose phosphate, hexosamine, nucleotide, and mevalonate pathways. Collectively, these findings expanded the repertoire of human GPI ligands and revealed previously unrecognized GPI-metabolite interactions that may contribute to modulation of glycolytic flux.

Macaranga tanarius (Euphorbiaceae), a myrmecophilic plant grown in subtropical countries, was identified as a source of Okinawan propolis, a honeybee product, which is glandular trichomes developed on the surface of the fruits. This tissue contains a high amount of characteristic geranylated flavonoids known as nymphaeols (nymphaeol A - C and isonymphaeol B), which exhibit strong antioxidant activity. In this study, we characterized the structure of glandular trichomes of M. tanarius, showing that they are sac-like structures containing drupelet-like aggregates of secretary cells, and a subcuticular cavity was also observed. From a cDNA library, we identified two prenyltransferase (PT) genes involved in biosynthesis of nymphaeols, and a PT gene grouped in a primary metabolic PT. Our biochemical experiments demonstrated that one of the former secondary metabolic PTs (MtPT1) is a B-ring-specific geranyltransferase for eriodictyol to yield nymphaeols B and isonymphaeol B. MtPT1 was unusual for a plant PT, an enzyme that yielded multiple reaction products. The other PT (MtPT3) transferred a geranyl moiety to the A-ring of eriodictyol to form nymphaeol A. Although dimethylallyl diphosphate (DMAPP) was not recognized as its prenyl donor substrate of eriodictyol, giving a B-ring geranylated eriodyctiol as the prenyl acceptor substrate, this enzyme used DMAPP to attach the prenyl moiety to the A-ring and produced nymphaeol C. These data suggest that M. tanarius has evolved its PT function to produce a diverse range of prenylated flavonoids using a limited set of genes.

Enzymatic sulfation offers a green-chemistry route to the selective sulfation of phenolic and other hydroxylated compounds. Bacterial arylsulfate sulfotransferases (ASSTs) are particularly attractive biocatalysts because they use simple aryl sulfate donors, such as p-nitrophenyl sulfate (pNPS), instead of the complex and labile cofactor PAPS required by conventional sulfotransferases. However, their rational application and engineering remain limited because the molecular principles controlling substrate recognition, catalytically competent donor and acceptor positioning, covalent sulfo-enzyme formation, and sulfo transfer are not fully understood. In particular, it has remained unclear how conserved active-site histidines cooperate during catalysis and how active-site preorganization promotes efficient sulfo transfer. Here, we address these questions for the newly characterized ASST (ASTB) from Desulfitobacterium hafniense clade 6 (Dh_ASST_6) by combining X-ray crystallography, steady-state enzyme kinetics, and quantum mechanics/molecular mechanics (QM/MM) simulations. Crystal structures of Dh_ASST_6 in the apo state and donor-soaked state reveal a monomeric {beta}-propeller enzyme with a histidine-centered active site and directly identify His350 as the transient sulfo carrier. Kinetic analysis shows efficient turnover and supports a ping-pong bi-bi mechanism, consistent with formation of a covalent sulfo-enzyme intermediate. Steered QM/MM simulations of the second catalytic step show that sulfuryl transfer from sulfated His350 to the phenolic acceptor proceeds through a proton-coupled, multistep pathway in which His283 assists acceptor activation by proton transfer. Active-site preorganization precedes the main bond-reorganization event and generates a near-attack geometry in which acceptor alignment, proton transfer, and stabilization of the sulfo group by a conserved hydrogen-bonding network jointly promote sulfo transfer. Free-energy reconstruction gives a low barrier of approximately 9.6 kcal/ mol, in agreement with efficient enzymatic turnover. The structural, kinetic, and computational data support a complete catalytic cycle for Dh_ASST_6 based on a cooperative two-histidine mechanism. This model establishes catalytically competent acceptor positioning and preservation of the histidine-centered hydrogen-bonding network as key design principles for ASST engineering. These features provide concrete targets for expanding substrate scope and tuning donor or acceptor preference while maintaining efficient sulfuryl transfer.