Key Residues Research Articles

DipR, a GntR/FadR-family transcriptional repressor: regulatory mechanism and widespread distribution of the dip cluster for dipicolinic acid catabolism in bacteria.

Dipicolinic acid is an essential component of bacterial spores for stress resistance, which is released into the environment after spore germination. In a previous study, a dip gene cluster was found to be responsible for the catabolism of dipicolinic acid in Alcaligenes faecalis JQ135. However, the transcriptional regulatory mechanism remains unclear. The present study characterized the new GntR/FadR family transcriptional factor DipR, showing that the dip cluster is transcribed as the six transcriptional units, dipR, dipA, dipBC, dipDEFG, dipHand dipJKLM. The purified DipR protein has six binding sites sharing the 6-bp conserved motif sequence 5'-GWATAC-3'. Site-directed mutations indicated that these motif sequences are essential for DipR binding. Moreover, the four key amino acid residues R63, R67, H196and H218 of DipR, examined by site-directed mutagenesis, played crucial roles in DipR regulation. Bioinformatics analysis showed that dip clusters including dipR genes are widely distributed in bacteria, are taxon-related, and co-evolved with their hosts. This paper provides new insights into the transcriptional regulatory mechanism of dipicolinic acid degradation by DipR in bacteria.

Synthesis and Biological Evaluation of Novel Piperidine-3-Carboxamide Derivatives as Anti-Osteoporosis Agents Targeting Cathepsin K.

A series of novel piperidamide-3-carboxamide derivatives were synthesized and evaluated for their inhibitory activities against cathepsin K. Among these derivatives, compound H-9 exhibited the most potent inhibition, with an IC50 value of 0.08 µM. Molecular docking studies revealed that H-9 formed several hydrogen bonds and hydrophobic interactions with key active-site residues of cathepsin K. In vitro, H-9 demonstrated anti-bone resorption effects that were comparable to those of MIV-711, a cathepsin K inhibitor currently in phase 2a clinical trials for the treatment of bone metabolic disease. Western blot analysis confirmed that H-9 effectively downregulated cathepsin K expression in RANKL-reduced RAW264.7 cells. Moreover, in vivo experiments showed that H-9 increased the bone mineral density of OVX-induced osteoporosis mice. These results suggest that H-9 is a potent anti-bone resorption agent targeting cathepsin K and warrants further investigation for its potential anti-osteoporosis values.

Hook loop dynamics engineering transcended the barrier of activity-stability trade-off and boosted the thermostability of enzymes

The improvement of enzyme thermostability often accompanies the decreased activity due to the loss of the key regions' flexibility. As a representative structure, unlocking the potential of loop dynamics will not only provide new ideas for stabilization strategies, but also help to deepen the understanding of the relationship between enzyme structural dynamics and function. In this study, a creative “hook loop dynamics engineering” (HLoD) strategy was successfully proposed for simultaneously improving the thermostability and maintaining activity of the model enzyme, Candida Antarctica lipase B. A small and smart mutant library involving five key residues located at the “hook loop” was meticulously identified and systematically investigated and thus yielded a five-point multiple mutant M1 (L147S/T244P/S250P/T256D/N292D), demonstrating a remarkable 7.0-fold increase in thermostability at 60 °C compared to the wild-type (WT). Furthermore, the activity of M1 remained comparable to that of WT, effectively transcending the barrier of activity-stability trade-off. Molecular dynamics simulations revealed that the precise regulation of hook loop dynamics via intermolecular interactions, such as salt bridges and hydrogen bonding, curbed the excessive flexibility of the pivotal regions α5 and α10 at high temperatures, thus driving the substantial enhancement of the thermostability of M1. Refining the dynamics of the flexible region via HLoD, which transcended the barrier of activity-stability trade-off, exhibited to be a robust and potentially universal strategy for designing enzymes with outstanding thermostability and activity.

Enhancing the apo protein tyrosine phosphatase non-receptor type 2 crystal soaking strategy through inhibitor-accessible binding sites.

Protein tyrosine phosphatase non-receptor type 2 (PTPN2) has recently been recognized as a promising target for cancer immunotherapy. Despite extensive structural and functional studies of other protein tyrosine phosphatases, there is limited structural understanding of PTPN2. Currently, there are only five published PTPN2 structures and none are truly unbound due to the presence of a mutation, an inhibitor or a loop (related to crystal packing) in the active site. In this report, a novel crystal packing is revealed that resulted in a true apo PTPN2 crystal structure with an unbound active site, allowing the active site to be observed in a native apo state for the first time. Key residues related to accommodation in the active site became identifiable upon comparison with previously published PTPN2 structures. Structures of PTPN2 in complex with an established PTPN1 active-site inhibitor and an allosteric inhibitor were achieved through soaking experiments using these apo PTPN2 crystals. The increased structural understanding of apo PTPN2 and the ability to soak in inhibitors will aid the development of future PTPN2 inhibitors.

ACAD10 and ACAD11 allow entry of 4-hydroxy fatty acids into β-oxidation

Hydroxylated fatty acids are important intermediates in lipid metabolism and signaling. Surprisingly, the metabolism of 4-hydroxy fatty acids remains largely unexplored. We found that both ACAD10 and ACAD11 unite two enzymatic activities to introduce these metabolites into mitochondrial and peroxisomal β-oxidation, respectively. First, they phosphorylate 4-hydroxyacyl-CoAs via a kinase domain, followed by an elimination of the phosphate to form enoyl-CoAs catalyzed by an acyl-CoA dehydrogenase (ACAD) domain. Studies in knockout cell lines revealed that ACAD10 preferentially metabolizes shorter chain 4-hydroxy fatty acids than ACAD11 (i.e. 6 carbons versus 10 carbons). Yet, recombinant proteins showed comparable activity on the corresponding 4-hydroxyacyl-CoAs. This suggests that the localization of ACAD10 and ACAD11 to mitochondria and peroxisomes, respectively, might influence their physiological substrate spectrum. Interestingly, we observed that ACAD10 is cleaved internally during its maturation generating a C-terminal part consisting of the ACAD domain, and an N-terminal part comprising the kinase domain and a haloacid dehalogenase (HAD) domain. HAD domains often exhibit phosphatase activity, but negligible activity was observed in the case of ACAD10. Yet, inactivation of a presumptive key residue in this domain significantly increased the kinase activity, suggesting that this domain might have acquired a regulatory function to prevent accumulation of the phospho-hydroxyacyl-CoA intermediate. Taken together, our work reveals that 4-hydroxy fatty acids enter mitochondrial and peroxisomal fatty acid β-oxidation via two enzymes with an overlapping substrate repertoire.

Functional Identification of the Isopentenyl Diphosphate Isomerase Gene from Fritillaria unibracteata

Isopentenyl diphosphate isomerase (IPI) is a key enzyme in the synthesis of isoprenoids. In this paper, the in vivo biological activity of the IPI gene from Fritillaria unibracteata (FuIPI) was investigated. Combining a color complementation experiment with High-Performance Liquid Chromatography analysis showed that the FuIPI gene could accumulate β-carotene in Escherichia coli, and Glu190 was identified as a key residue for its catalytic activity. Bioinformatics analysis together with subcellular localization indicated that the FuIPI protein was localized in chloroplasts. Compared with wild-type Arabidopsis thaliana, FuIPI transgenic plants had higher abscisic acid content and strengthening tolerance to drought and salt stress. Overall, these results indicated that the FuIPI gene had substantial biological activity in vivo, hopefully laying a foundation for its further research and application in liliaceous ornamental and medicinal plants.

Volatile profiling of Cinnamomum heyneanum and Cinnamomum Palghatensis and in vitro and in silico antidiabetic activity of essential oil nanoemulsions

Essential oil nanoemulsions (EONs) of two wild cinnamons, Cinnamomum heyneanum (CH) and Cinnamomum Palghatensis (CP), were formulated, characterized and investigated for their antidiabetic activity. Leaf essential oils (EOs) were extracted and characterized by GC-MS and GC-FID. Two sets of EONs were formulated and characterized by zeta analyzer, viscometer and TEM. Antioxidant activity was studied using DPPH, ABTS, superoxide, hydroxyl and hydrogen peroxide assays. Antidiabetic activity was evaluated by α-amylase inhibition, α-glucosidase inhibition, glucose uptake and glucose-stimulated insulin secretion assays. In silico studies were performed to support the efficacy. Methyl eugenol (60.3 %), safrole (27.8 %) in CH EO and bicyclogermacrene (19.5 %), (E)-caryophyllene (14 %) in CP EO were major components. Of two sets of EONs, 1:2 ratio showed better hydrodynamic average diameter [CH 42.9±0.78 nm & CP 27.9±1.70 nm] and better zeta potential [CH −38.4±0.85 mV & CP −38.4±0.45 mV (1st day)]. TEM observation showed that 1:2 ratio EONs were spherical with an average diameter of 85 nm (CH) and 8.98 nm (CP). Both CH and CP showed remarkable antioxidant activity. CP showed better α-glucosidase inhibition (IC50 0.599±0.008 mg/mL), enhanced glucose uptake (0.53±0.08 fold vs. control) and insulin secretion (1.40±0.06 fold vs. control). In silico analysis reveals that all major components of CH and CP interact with key residues of the protein targets associated with diabetes, α-amylase, α-glucosidase, insulin receptor, Glugagon like peptide1 Receptor and Glut 4. In vitro and in silico studies revealed that EON of CH and CP have pharmacological properties due to the presence of various compounds against diabetes mellitus.

Mechanism of the interaction between olfactory receptors and characteristic aroma compounds in sweet orange juice

The aroma compounds of three varieties of orange juice were extracted using solvent-assisted flavor evaporation (SAFE) and stir bar sorptive extraction (SBSE). A total of 41 aroma compounds were identified by gas chromatography-olfactometry (GC-O), among which 10 aroma compounds had an aroma intensity (AI) > 4.5. There were 53 aroma compounds identified by gas chromatography-mass spectrometry (GC-MS), 29 of which had an odor activity value (OAV) higher than 1. The findings indicated that (+)-limonene, linalool, hexanal, citral and myrcene were closely associated with the five characteristic aromas of orange juice. Furthermore, molecular docking was used to analyze the binding energies and interactions between these compounds and olfactory receptors (OR1A1, OR1D2, OR2W1, OR5AC2, OR1A2, and OR5M3). The average binding energies for these compounds at the six receptors were −6.2 kcal/mol, −6.0 kcal/mol, −5.8 kcal/mol, −5.1 kcal/mol, −6.3 kcal/mol and −4.7 kcal/mol, respectively. Hydrophobic interaction was a significant driving force in this process. Key residues such as Leu199 (OR1D2), Ala108 (OR1A2), Gly108 (OR2W1), Pro181 (OR5M3), Tyr261 (OR5AC2), and Val203 (OR1A1) played key roles in binding with characteristic aroma compounds. The formation mechanism of orange juice aroma was revealed, which provides theoretical guidance for the regulation of sweet orange aroma and flavor.

Unveiling the potential of phytochemicals to inhibit nuclear receptor binding SET domain protein 2 for cancer: Pharmacophore screening, molecular docking, ADME properties, and molecular dynamics simulation investigations.

Nuclear receptor binding SET domain protein 2 (NSD2) significantly contributes to the development of cancer, making it a promising target for cancer drug discovery. This research explores natural compounds as potential selective inhibitors for NSD2 in cancer treatment. Employing a comprehensive in silico approach, the study utilized pharmacophore modeling, molecular docking, pharmacokinetic profiling, and molecular dynamics simulations. An e-pharmacophore model-based screening using the first selective and potent ligand bound to NSD2 identified 49,248 natural compounds from the SuperNatural 3.0 database (containing 449,008 molecules) with acceptable alignment with the developed pharmacophore hypotheses. Subsequently, molecular docking was executed to assess the standout compounds which led to the selection of ten candidates that surpassed the reference inhibitor in accordance w the binding affinity expressed as a G score. Ligand-residue interaction analyses of the top three hits (SN0450102, SN0410255, and SN0142336) revealed diverse crucial interactions with the NSD2 active site, including hydrogen bonds, pi-pi stacking, and hydrophobic contacts with key amino acid residues in the NSD2-PWWP1 domain. Pharmacokinetic profiling confirmed the drug-likability for the refined hits, indicating good cellular permeability and minimal blood-brain barrier penetration. Molecular dynamics simulations for 200 nanoseconds affirmed the stability of protein-ligand complexes, with minimal fluctuations in root mean square deviation and root mean square fluctuation analyses. Overall, this study identified promising natural compounds as potential pharmaceutical agents in the treatment of NSD2-associated cancers.

Alisol C 23-acetate might be a lead compound of potential lipase inhibitor from Alismatis Rhizoma: Screening, identification and molecular dynamics simulation

Alismatis Rhizoma (AR), a traditional Chinese medicine for treating obesity in traditional Chinese medicine clinic, is recognized as a promising source of lead compounds of lipase inhibitors. Ultrafiltration centrifugal combined with liquid chromatography-mass spectrometry (UF-LC-MS) was used for screening potential lipase inhibitors from AR, and the result indicated the binding capacity between compound 7 and lipase (92.3 ± 1.28 %) was significantly higher than other triterpenoids, and was identified as alisol C 23-acetate. It exhibited a mixed-type inhibitory behavior with an IC50 value of 84.88 ± 1.03 μM. Subsequently, the binding pockets of alisol C 23-acetate to lipase were predicted, and their binding mechanism was explored with molecular simulation. Pocket 1 (active center) and pocket 4 might be the orthosteric and allosteric binding sites of alisol C 23-acetate to lipase, respectively. The interaction between alisol C 23-acetate and lipase was identified to involve key amino acid residues such as GLY-77, PHE-78, TYR-115, LEU-154, PRO-181, PHE-216, LEU-264, ASP-278, GLN-306, ARG-313, and VAL-426. Meanwhile, alisol C 23-acetate remained stable during the intestinal digestive but degraded in the gastric digestion. Overall, alisol C 23-acetate is expected to be the lead compound of lipase inhibitors for treating obesity.

GPRC6A is a Potential Therapeutic Target for Metformin Regulation of Glucose Homeostasis in Mice.

Understanding the mechanism of metformin actions in treating type 2 diabetes is limited by an incomplete knowledge of the specific protein targets mediating its metabolic effects. Metformin has structural similarities to L-Arginine (2-amino-5-guanidinopentanoic acid), which is a ligand for GPRC6A, a Family C G-protein coupled receptor that regulates energy metabolism. Ligand activation of GPRC6A results in lowering of blood glucose and other metabolic changes resembling the therapeutic effect of metformin. In the current study, we tested if metformin activates GPRC6A. We used Alphafold2 to develop a structural model for L-Arginine (L-Arg) binding to the extracellu-lar bilobed venus flytrap domain (VFT) of GPRC6A. We found that metformin docked to the site in the VFT that overlaps the binding site for L-Arg. Metformin resulted in a dose-dependent stimulation of GPRC6A activity in HEK-293 cells transfected with full-length wild-type GPRC6A but not in untransfected control cells. In addition, metformin failed to activate an alternatively spliced GPRC6A isoform lacking the putative binding site in the VFT. More specifically, mutation of the predicted metformin key binding residues Glu170 and Asp303 in the GPRC6A VFT resulted in loss of metformin receptor activation in vitro. The in vivo role of GPRC6A in mediating the effects of metformin was tested in Gprc6a-/- mice. Administration of therapeutic doses of metformin lowered blood glucose levels following a glucose tolerance test in wild-type but not Gprc6a-/- mice. Finally, we EN300, created by adding a carboxymethyl group from L-Arg to the biguanide backbone of metformin. EN300 showed dose-dependent stimulation of GPRC6A activity in vitro with greater potency than L-Arginine, but less than metformin. Thus, we suggest that GPRC6A is a potential molecular target for metformin which may be used to understand the therapeutic actions of metformin and develop novel small molecules to treat T2D.

Structure and dimerization properties of the plant-specific copper chaperone CCH

Copper chaperones of the ATX1 family are found in a wide range of organisms where these essential soluble carriers strictly control the transport of monovalent copper across the cytoplasm to various targets in diverse cellular compartments thereby preventing detrimental radical formation catalyzed by the free metal ion. Notably, the ATX1 family in plants contains two distinct forms of the cellular copper carrier. In addition to ATX1 having orthologs in other species, they also contain the copper chaperone CCH. The latter features an extra C-terminal extension whose function is still unknown. The secondary structure of this extension was predicted to be disordered in previous studies, although this has not been experimentally confirmed. Solution NMR studies on purified CCH presented in this study disclose that this region is intrinsically disordered regardless of the chaperone’s copper loading state. Further biophysical analyses of the purified metallochaperone provide evidence that the C-terminal extension stabilizes chaperone dimerization in the copper-free and copper-bound states. A variant of CCH lacking the C-terminal extension, termed CCHΔ, shows weaker dimerization but similar copper binding. Computational studies further corroborate the stabilizing role of the C-terminal extension in chaperone dimerization and identify key residues that are vital to maintaining dimer stability.

Identification of small molecule inhibitors of the Chloracidobacterium thermophilum type IV pilus protein PilB by ensemble virtual screening

Antivirulence strategy has been explored as an alternative to traditional antibiotic development. The bacterial type IV pilus is a virulence factor involved in host invasion and colonization in many antibiotic resistant pathogens. The PilB ATPase hydrolyzes ATP to drive the assembly of the pilus filament from pilin subunits. We evaluated Chloracidobacterium thermophilum PilB (CtPilB) as a model for structure-based virtual screening by molecular docking and molecular dynamics (MD) simulations. A hexameric structure of CtPilB was generated through homology modeling based on an existing crystal structure of a PilB from Geobacter metallireducens. Four representative structures were obtained from molecular dynamics simulations to examine the conformational plasticity of PilB and improve docking analyses by ensemble docking. Structural analyses after 1 μs of simulation revealed conformational changes in individual PilB subunits are dependent on ligand presence. Further, ensemble virtual screening of a library of 4234 compounds retrieved from the ZINC15 database identified five promising PilB inhibitors. Molecular docking and binding analyses using the four representative structures from MD simulations revealed that top-ranked compounds interact with multiple Walker A residues, one Asp-box residue, and one arginine finger, indicating these are key residues in inhibitor binding within the ATP binding pocket. The use of multiple conformations in molecular screening can provide greater insight into compound flexibility within receptor sites and better inform future drug development for therapeutics targeting the type IV pilus assembly ATPase.

Structural insight into the function of human peptidyl arginine deiminase 6

Peptidyl arginine deiminase 6 (PADI6 or PAD6) is vital for early embryonic development in mice and humans, yet its function remains elusive. PADI6 is less conserved than other PADIs and it is currently unknown whether it has a catalytic function. Here we show that human PADI6 dimerises like hPADIs 2–4, however, does not bind Ca2+ and is inactive in in vitro assays against standard PADI substrates. By determining the crystal structure of hPADI6, we show that hPADI6 is structured in the absence of Ca2+ where hPADI2 and hPADI4 are not, and the Ca-binding sites are not conserved. Moreover, we show that whilst the key catalytic aspartic acid and histidine residues are structurally conserved, the cysteine is displaced far from the active site centre and the hPADI6 active site pocket appears closed through a unique evolved mechanism in hPADI6, not present in the other PADIs. Taken together, these findings provide insight into how the function of hPADI6 may differ from the other PADIs based on its structure and provides a resource for characterising the damaging effect of clinically significant PADI6 variants.

Molecular insight into binding affinities and blockade effects of selected flavonoid compounds on the PD-1/PD-L1 pathway.

This study investigated the binding mechanisms of the flavonoids apigenin (Api), kaempferol (Kmp), and quercetin (Que) to the PD-L1 dimer using a combination of molecular modeling and experimental techniques. The binding free energy results demonstrated that the flavonoids could tightly bind to the PD-L1 dimer, with the binding abilities following the trend Que > Kmp > Api. Key residues Ile54, Tyr56, Met115, Ala121, and Tyr123 were identified as important for binding. The flavonoids primarily bind to the C-, F-, and G-sheet domains. The spontaneous formation of the complex systems was mainly driven by hydrophobic forces. Dynamic cross-correlation matrix and secondary structure analyses further indicated that the studied flavonoids could stably interact with the binding sites. ELISA results showed that the flavonoids could effectively block PD-1/PD-L1 interactions, although the inhibitory activity of Api was weaker. Therefore, flavonols might be more effective inhibitors compared to flavones. The findings of this study are expected to contribute to the development of novel flavonoids targeting the PD-1/PD-L1 pathway.

Structural analysis of the human C5a-C5aR1 complex using cryo-electron microscopy

The complement system is a complex network of proteins that plays a crucial role in the innate immune response. One important component of this system is the C5a-C5aR1 complex, which is critical in the recruitment and activation of immune cells. In-depth investigation of the activation mechanism as well as biased signaling of the C5a-C5aR1 system will facilitate the elucidation of C5a-mediated pathophysiology. In this study, we determined the structure of C5a-C5aR1-Gi complex at a high resolution of 3 Å using cryo-electron microscopy (Cryo-EM). Our results revealed the binding site of C5a, which consists of a polar recognition region on the extracellular side and an amphipathic pocket within the transmembrane domain. Furthermore, we found that C5a binding induces conformational changes of C5aR1, which subsequently leads to the activation of G protein signaling pathways. Notably, a key residue (M265) located on transmembrane helix 6 (TM6) was identified to play a crucial role in regulating the recruitment of β-arrestin driven by C5a. This study provides more information about the structure and function of the human C5a-C5aR1 complex, which is essential for the proper functioning of the complement system. The findings of this study can also provide a foundation for the design of new pharmaceuticals targeting this receptor with bias or specificity.

The effect of phosphorylation efficiency on the oncogenic properties of the protein E7 from high-risk HPV

The Human papillomavirus (HPV) causes tumors in part by hijacking the host cell cycle and forcing uncontrolled cellular division. While there are >200 genotypes of HPV, 15 are classified as high-risk and have been shown to transform infected cells and contribute to tumor formation. The remaining low-risk genotypes are not considered oncogenic and result in benign skin lesions. In high-risk HPV, the oncoprotein E7 contributes to the dysregulation of cell cycle regulatory mechanisms. High-risk E7 is phosphorylated in cells at two conserved serine residues by Casein Kinase 2 (CK2) and this phosphorylation event increases binding affinity for cellular proteins such as the tumor suppressor retinoblastoma (pRb). While low-risk E7 possesses similar serine residues, it is phosphorylated to a lesser degree in cells and has decreased binding capabilities. When E7 binding affinity is decreased, it is less able to facilitate complex interactions between proteins and therefore has less capability to dysregulate the cell cycle. By comparing E7 protein sequences from both low- and high-risk HPV variants and using site-directed mutagenesis combined with NMR spectroscopy and cell-based assays, we demonstrate that the presence of two key nonpolar valine residues within the CK2 recognition sequence, present in low-risk E7, reduces serine phosphorylation efficiency relative to high-risk E7. This results in significant loss of the ability of E7 to degrade the retinoblastoma tumor suppressor protein, thus also reducing the ability of E7 to increase cellular proliferation and reduce senescence. This provides additional insight into the differential E7-mediated outcomes when cells are infected with high-risk verses low-risk HPV. Understanding these oncogenic differences may be important to developing targeted treatment options for HPV-induced cancers.

Biochemical and structural elucidation of the L-carnitine degradation pathway of the human pathogen Acinetobacter baumannii.

Acinetobacter baumannii is an opportunistic human pathogen which can use host-derived L-carnitine as sole carbon and energy source. Recently, an L-carnitine transporter (Aci1347) and a specific monooxygense (CntA/CntB) for the intracellular cleavage of L-carnitine have been characterized. Subsequent conversion of the resulting malic semialdehyde into the central metabolite L-malate was hypothesized. Alternatively, L-carnitine degradation via D-malate with subsequent oxidation into pyruvate was proposed. Here we describe the in vitro and in vivo reconstitution of the entire pathway, starting from the as yet uncharacterized gene products of the carnitine degradation gene operon. Using recombinantly purified enzymes, enantiomer-specific formation of D-malate by the NAD(P)+-dependent malic semialdehyde dehydrogenase (MSA-DH) is demonstrated. The solved X-ray crystal structure of tetrameric MSA-DH reveals the key catalytic residues Cys290 and Glu256, accessible through opposing substrate and cofactor funnels. Specific substrate binding is enabled by Arg166, Arg284 and Ser447 while dual cofactor specificity for NAD+ and NADP+ is mediated by Asn184. The subsequent conversion of the unusual D-malate reaction product by an uncharacterized NAD+-dependent malate dehydrogenase (MDH) is shown. Tetrameric MDH is a β-decarboxylating dehydrogenase that synthesizes pyruvate. MDH experiments with alternative substrates showed a high degree of substrate specificity. Finally, the entire A. baumannni pathway was heterologously reconstituted, allowing E. coli to grow on L-carnitine as a carbon and energy source. Overall, the metabolic conversion of L-carnitine via malic semialdehyde and D-malate into pyruvate, CO2 and trimethylamine was demonstrated. Trimethylamine is also an important gut microbiota-dependent metabolite that is associated with an increased risk of cardiovascular disease. The pathway reconstitution experiments allowed us to assess the TMA forming capacity of gut microbes which is related to human cardiovascular health.

Identification of a new retigabine derivative with improved photostability for selective activation of neuronal Kv7 channels and antiseizure activity.

Pharmacological activation of neuronal Kv7 channels by the antiepileptic drug retigabine (RTG; ezogabine) has been proven effective in treating partial epilepsy. However, RTG was withdrawn from the market due to the toxicity caused by its phenazinium dimer metabolites, leading to peripheral skin discoloration and retinal abnormalities. To address the undesirable metabolic properties of RTG and prevent the formation of phenazinium dimers, we made chemical modifications to RTG, resulting in a new RTG derivative, 1025c, N,N'-{4-[(4-fluorobenzyl) (prop-2-yn-1-yl)amino]-1,2-phenylene}bis(3,3-dimethylbutanamide). Whole-cell recordings were used to evaluate Kv7 channel openers. Site-directed mutagenesis and molecular docking were adopted to investigate the molecular mechanism underlying 1025c and Kv7.2 interactions. Mouse seizure models of maximal electroshock (MES), subcutaneous pentylenetetrazol (scPTZ), and PTZ-induced kindling were utilized to test compound antiepileptic activity. The novel compound 1025c selectively activates whole-cell Kv7.2/7.3 currents in a concentration-dependent manner, with half-maximal effective concentration of .91 ± .17 μmol·L-1. The 1025c compound also causes a leftward shift in Kv7.2/7.3 current activation toward a more hyperpolarized membrane potential, with a shift of the half voltage of maximal activation (ΔV1/2) of -18.6 ± 3.0 mV. Intraperitoneal administration of 1025c demonstrates dose-dependent antiseizure activities in assays of MES, scPTZ, and PTZ-induced kindling models. Moreover, through site-directed mutagenesis combined with molecular docking, a key residue Trp236 has been identified as critical for 1025c-mediated activation of Kv7.2 channels. Photostability experiments further reveal that 1025c is more photostable than RTG and is unable to dimerize. Our findings demonstrate that 1025c exhibits potent and selective activation of neuronal Kv7 channels without being metabolized to phenazinium dimers, suggesting its developmental potential as an antiseizure agent for therapy.

Revisiting legume lectins: Structural organization and carbohydrate-binding properties

Legume lectins are a diverse family of carbohydrate-binding proteins that share significant similarities in their primary, secondary, and tertiary structures, yet exhibit remarkable variability in their quaternary structures and carbohydrate-binding specificities. The tertiary structure of legume lectins, characterized by a conserved β-sandwich fold, provides the scaffold for the formation of a carbohydrate-recognition domain (CRD) responsible for ligand binding. The structural basis for the binding is similar between members of the family, with key residues interacting with the sugar through hydrogen bonds, hydrophobic interactions, and van der Waals forces. Variability in substructures and residues within the CRD are responsible for the large array of specificities and enable legume lectins to recognize diverse sugar structures, while maintaining a consistent structural fold. Therefore, legume lectins can be classified into several specificity groups based on their preferred ligands, including mannose/glucose-specific, N-acetyl-d-galactosamine/galactose-specific, N-acetyl-d-glucosamine-specific, l-fucose-specific, and α-2,3 sialic acid-specific lectins. In this context, this review examined the structural aspects and carbohydrate-binding properties of representative legume lectins and their specific ligands in detail. Understanding the structure/binding relationships of lectins continues to provide valuable insights into their biological roles, while also assisting in the potential applications of these proteins in glycobiology, diagnostics, and therapeutics.