Recombinant Enzyme Research Articles

Biochemical characterization of a novel bifunctional alginate lyase from Microbulbifer arenaceous

Bio‒production of alginate oligosaccharides (AOSs), a type of functional food additive, is a promising way for green utilization of algae, in which alginate hydrolyzing enzymes play a key role. A novel alginate lyase gene (MiAly17A) from a marine bacterium Microbulbifer arenaceous was heterologously expressed in E. coli. The coding sequence of the gene shared the highest identity of 86% with a polysaccharide lyase (PL) family 17 alginate lyase (AlgL17) from Microbulbifer sp. ALW1. The recombinant enzyme (MiAly17A) was purified and biochemically characterized. MiAly17A showed maximal enzyme activity at 40 °C and pH 7.5, respectively. It was stable at the temperatures below 35 °C and within pH 5.0–8.0. The enzyme activities were increased by 5.3 and 5.6 folds in the presence of 100 mM of K+ and Na+, respectively. MiAly17A was bifunctional and could hydrolyze sodium alginate to release unsaturated monosaccharides and oligosaccharides with degrees of polymerization (DP) 2–7. The enzyme catalyzed the cleavage of glycosidic bonds from the non-reducing ends and the backbone of the tested oligosaccharides (DP ≥ 4), exhibiting both exolytic and endo-lytic activities. Moreover, MiAly17A was used for the production of alginate oligosaccharides from sodium alginate, and the highest conversion ratio of 68% was obtained. The unique properties may possess the enzyme great potential for preparation of alginate oligosaccharides.

In Search of Complementary Extraction Methods for Comprehensive Coverage of the Escherichia coli Metabolome.

Escherichia coli is an invaluable research tool for many fields of biology, in particular for the production of recombinant enzymes. However, the activity of many such recombinant enzymes cannot be determined using standard biochemical assays, as often, the relevant substrates are not known, or the products produced are not detectable. Today, the biochemical footprints of such unknown enzyme activities can be revealed via the analysis of the metabolomes of the recombinant E. coli clones in which they are expressed, using sensitive technologies such as mass spectrometry. However, before any metabolites can be identified, it is necessary to achieve as high a coverage of the potential metabolites present within E. coli as possible. We have therefore analyzed a wide range of different extraction methods against the cell free extracts of various recombinant E. coli clones. The results were analyzed to determine the minimum number of extractions that achieved high recovery and coverage of metabolites. Two methods were selected for further analysis due to their ability to produce not only high numbers of ions, but also wide mass coverage and a high degree of complementarity. One extraction method uses acetonitrile and water, in a 4:1 ratio, which is then dried down and reconstituted in the chromatography running buffer prior to injection onto the chromatography column, and the other extraction method uses a combination of methanol, water and chloroform, in a 3:1:1 ratio, which is injected directly onto the chromatography column. These two extraction methods were shown to be complementary to each other, as regards the respective metabolites extracted, and to cover a large range of metabolites.

A cell-free multi-enzyme cascade for the synthesis of CDP-glycerol.

CDP-glycerol is a nucleotide-diphosphate-activated version of glycerol. In nature, it is required for the biosynthesis of teichoic acid in Gram-positive bacteria, which is an appealing target epitope for the development of new vaccines. Here, a cell-free multi-enzyme cascade was developed to synthetize nucleotide-activated glycerol from the inexpensive and readily available substrates cytidine and glycerol. The cascade comprises five recombinant enzymes expressed in E. coli that were purified by immobilized metal affinity chromatography. As part of the cascade, ATP is in-situ regenerated from polyphosphate to reduce synthesis costs. The enzymatic cascade was characterized at laboratory scale and the products analyzed by high performance anion exchange chromatography (HPAEC)-UV and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). After the successful synthesis was confirmed, a design of experiments approach was used to screen for optimal operation conditions (temperature, pH value and MgCl2 concentration). Overall, a substrate conversion of 89% was achieved with respect to the substrate cytidine.

Enzyme Engineering Strategies for the Bioenhancement of L-Asparaginase Used as a Biopharmaceutical.

Over the past few years, there has been a surge in the industrial production of recombinant enzymes from microorganisms due to their catalytic characteristics being highly efficient, selective, and biocompatible. L-asparaginase (L-ASNase) is an enzyme belonging to the class of amidohydrolases that catalyzes the hydrolysis of L-asparagine into L-aspartic acid and ammonia. It has been widely investigated as a biologic agent for its antineoplastic properties in treating acute lymphoblastic leukemia. The demand for L-ASNase is mainly met by the production of recombinant type II L-ASNase from Escherichia coli and Erwinia chrysanthemi. However, the presence of immunogenic proteins in L-ASNase sourced from prokaryotes has been known to result in adverse reactions in patients undergoing treatment. As a result, efforts are being made to explore strategies that can help mitigate the immunogenicity of the drug. This review gives an overview of recent biotechnological breakthroughs in enzyme engineering techniques and technologies used to improve anti-leukemic L-ASNase, taking into account the pharmacological importance of L-ASNase.

Structural and biochemical characterisation of the N-carbamoyl-β-alanine amidohydrolase from Rhizobium radiobacter MDC 8606.

N-carbamoyl-β-alanine amidohydrolase (CβAA) constitutes one of the most important groups of industrially relevant enzymes used in the production of optically pure amino acids and derivatives. In this study, a CβAA-encoding gene from Rhizobium radiobacter strain MDC 8606 was cloned and overexpressed in Escherichia coli. The purified recombinant enzyme (RrCβAA) showed a specific activity of 14 U·mg-1 using N-carbamoyl-β-alanine as a substrate with an optimum activity at 55 °C and pH 8.0. In this work, we report also the first prokaryotic CβAA structure at a resolution of 2.0 Å. A discontinuous catalytic domain and a dimerisation domain attached through a flexible hinge region at the domain interface have been revealed. We identify key ligand binding residues, including a conserved glutamic acid (Glu131), histidine (H385) and arginine (Arg291). Our results allowed us to explain the preference of the enzyme for linear carbamoyl substrates, as large and branched carbamoyl substrates cannot fit in the active site of the enzyme. This work envisages the use of RrCβAA from R. radiobacter MDC 8606 for the industrial production of L-α-, L-β- and L-γ-amino acids. The structural analysis provides new insights on enzyme-substrate interaction, which shed light on engineering of CβAAs for high catalytic activity and broad substrate specificity.

Elucidation of the mescaline biosynthetic pathway in peyote (Lophophora williamsii).

Peyote (Lophophora williamsii) is an entheogenic and medicinal cactus native to the Chihuahuan desert. The psychoactive and hallucinogenic properties of peyote are principally attributed to the phenethylamine derivative mescaline. Despite the isolation of mescaline from peyote over 120 years ago, the biosynthetic pathway in the plant has remained undiscovered. Here, we use a transcriptomics and homology-guided gene discovery strategy to elucidate a near-complete biosynthetic pathway from l-tyrosine to mescaline. We identified a cytochrome P450 that catalyzes the 3-hydroxylation of l-tyrosine to l-DOPA, a tyrosine/DOPA decarboxylase yielding dopamine, and four substrate-specific and regiospecific substituted phenethylamine O-methyltransferases. Biochemical assays with recombinant enzymes or functional analyses performed by feeding putative precursors to engineered yeast (Saccharomyces cerevisiae) strains expressing candidate peyote biosynthetic genes were used to determine substrate specificity, which served as the basis for pathway elucidation. Additionally, an N-methyltransferase displaying broad substrate specificity and leading to the production of N-methylated phenethylamine derivatives was identified, which could also function as an early step in the biosynthesis of tetrahydroisoquinoline alkaloids in peyote.

Improved anti-biofilm activity and long-lasting effects of novel serratiopeptidase immobilized on cellulose nanofibers.

Today, enzymatic treatment is a progressive field in combating biofilm producing pathogens. In this regard, serratiopeptidase, a medicinally important metalloprotease, has been recently highlighted as an enzyme with proved anti-biofilm activity. In the present study, in order to increase the long-lasting effects of the enzyme, serratiopeptidase and the novel engineered forms with enhanced anti-biofilm activity were immobilized on the surface of cellulose nanofibers (CNFs) as a natural polymer with eminent properties. For this, recombinant serratiopeptidases including the native and previously designed enzymes were produced, purified and conjugated to the CNF by chemical and physical methods. Immobilization was confirmed using different scanning and microscopic methods. The enzyme activity was assessed using casein hydrolysis test. Enzyme release analysis was performed using dialysis tube method. Anti-biofilm activity of free and immobilized enzymes has been examined on Staphylococcus aureus and Pseudomonas aeruginosa strains. Finally, cytotoxicity of enzyme-conjugated CNFs was performed by MTT assay. The casein hydrolysis results confirmed fixation of all recombinant enzymes on CNFs by chemical method; however, inadequate fixation of these enzymes was found using cold atmospheric plasma (CAP). The AFM, FTIR, and SEM analysis confirmed appropriate conjugation of enzymes on the surface of CNFs. Immobilization of enzymes on CNFs improved the anti-biofilm activity of serratiopeptidase enzymes. Interestingly, the novel engineered serratiopeptidase (T344 [8-339ss]) exhibited the highest anti-biofilm activity in both conjugated and non-conjugated forms. In conclusion, incorporation of serratiopeptidases into CNFs improves their anti-biofilm activities without baring any cytotoxicity. KEY POINTS: • Enzymes were successfully immobilized on cellulose nanofibers using chemical method. • Immobilization of enzymes on CNFs improved their anti-biofilm activity. • T344 [8-339ss] exhibited the highest anti-biofilm activity in both conjugated and non-conjugated forms.

N, N-dimethyltryptamine forms oxygenated metabolites via CYP2D6 - an in vitro investigation

N, N-dimethyltryptamine (DMT) is a psychedelic compound that has shown potential in the treatment of depression. Aside from the primary role of monoamine oxidase A (MAO-A) in DMT metabolism, the metabolic pathways are poorly understood. Increasing this understanding is an essential aspect of ensuring safe and efficacious use of DMT. This work aimed to investigate the cytochrome 450 (CYP) mediated metabolism of DMT by incubating DMT with recombinant human CYP enzymes and human liver microsomes (HLM) followed by analysis using high-resolution mass spectrometry for metabolite identification. DMT was rapidly metabolised by CYP2D6, while stable with all other investigated CYP enzymes. The metabolism of DMT in HLM was reduced after inclusion of harmine and SKF-525A whereas quinidine did not affect the metabolic rate, likely due to MAO-A residues present in HLM. Analysis of the CYP2D6 incubates showed formation of mono-, di- and tri-oxygenated metabolites, likely as a result of hydroxylation on the indole core. More research is needed to investigate the role of this metabolic pathway in vivo and any pharmacological activity of the proposed metabolites. Our findings may impact on safety issues following intake of ayahuasca in slow CYP2D6 metabolizers or with concomitant use of CYP2D6 inhibitors.

NAD+-Dependent Formate Dehydrogenase from Themotolerant Yeast Ogataea parapolymorpha: Properties and Protein Engineering of the N-Terminal Sequence.

Previously, the gene of formate dehydrogenase (FDH, EC 1.2.1.2) from the thermotolerant methylotrophic yeast Ogataea parapolymorpha DL 1 (OpaFDH) was cloned in our laboratory. Recombinant enzyme with additional glycine amino acid residue (OpaFDH_GK) was obtained in Escherichia coli cells in active and soluble form with a yield of more than 1 g per liter of the medium. In the present work, a detailed comparison of this enzyme with FDHs from other sources was carried out. Among eukaryotic formate dehydrogenases, OpaFDH has the highest thermal stability. To elucidate effect of N-terminal residue on the properties of the enzyme, OpaFDH_K (identical to natural) and OpaFDH_AK variants containing an additional Ala residue at the N-terminus were also obtained. It was shown that addition of an Ala residue to the N-terminus reduces four-fold the rate constant of thermal inactivation compared with the addition of a Gly residue. Addition of six more histidine residues to the N-terminus of OpaFDH_AK leads to acceleration of purification, practically does not affect kinetic parameters, but somewhat reduces thermal stability, which, however, can be restored to the level of OpaFDH_AK stability by adding 0.5 M NaCl.

Expression of Synthetic cyp102A1-LG23 Gene and Functional Analysis of Recombinant Cytochrome P450 BM3-LG23 in the Actinobacterium Mycolicibacterium smegmatis.

Cytochrome CYP102A1 (P450 BM3) of Priestia megaterium (bas. Bacillus megaterium) has several unique functional features and thus provides an ideal object for directed evolution and other synthetic applications. Previously, the CYP102A1-LG23 mutant with 14 mutations in the heme part was obtained that hydroxylates several androstanes at C7β with the formation of products with the anti-inflammatory and neuroprotective activities. In this study, synthetic cyp102A1-LG23 gene encoding the P450 BM3 mutant was expressed as a component of either monocistronic operon or bicistronic operon containing the gdh (glucose dehydrogenase, GDH) or zwf2 (glucose 6-phosphate dehydrogenase, G6PD) gene in Mycolicibacterium smegmatis BD cells. The recombinant bacteria were able hydroxylate androst-4-ene-3,17-dione (AD) into 7β-OH-AD. Their biocatalytic activity was increased twice by increasing the solubility of CYP102A1-LG23 protein in the cells and supplementing the cells with the additional cofactor regeneration system by introducing GDH and G6PD. The maximum 7β-OH-AD yield (37.68 mol%) was achieved by co-expression of cyp102A1-LG23 and gdh genes in M. smegmatis. These results demonstrate the possibility of using synthetic genes to obtain recombinant enzymes and expand our understanding of the processes involved in steroid hydroxylation by bacterial cytochromes. The data obtained can be used to develop new approaches for microbiological production of 7β-hydroxylated steroids in genetically modified Mycolicibacterium species.

Can velmanase alfa be the next widespread potential therapy for alpha-mannosidosis?

Alpha-mannosidosis (AM) is an autosomal recessive lysosomal storage disorder caused by reduced activity of the enzyme alpha-mannosidase. The disease is characterized by immunodeficiency, facial and skeletal abnormalities, impaired hearing, and intellectual disability. The clinical subtype of AM shows considerable variability in an individual, and at present, at least three clinical subtypes are suggested. Diagnosis is made by identification of deficiency of α-mannosidase activity in nucleated cells, like fibroblasts. The children are often born apparently normal as the disease is insidiously progressive, hence making early diagnosis essential. Along with supportive care, long-term therapeutic options include hematopoietic stem cell transplant, bone marrow transplantation, and enzyme replacement therapy. The possible benefits of these procedures must be weighed against the overall risk of procedure-related morbidity and mortality. Velmanase alfa is the first human recombinant form of alpha-mannosidase licensed and available for long-term enzyme replacement therapy. It is approved for treating non-neurologic manifestations of mild to moderate AM. The results obtained from different clinical trials provide evidence of the positive clinical effect of the recombinant enzyme on patients with AM. Different routes of diagnosis and unspecific initial symptoms of the disease lead to a delay in the initiation of treatment, resulting in accumulative morbidity. Thus, there is a dire necessity to create more awareness. Furthermore, additional multiple large-scale trials are needed to evaluate the long-term safety and efficacy of velmanase alfa.

Identification and Thermostability Modification of the Mesophilic L-asparaginase from Limosilactobacillus secaliphilus.

L-asparaginase (L-ASNase, E.C.3.5.1.1) could effectively inhibit the formation of acrylamide (AA) by hydrolyzing the AA precursor L-asparagine. However, most of the L-ASNases showed a relatively weak thermostability, posing a big threat on the application of enzyme at high processing temperatures. Here, the recombinant L-ASNase from mesophilic bacteria Limosilactobacillus secaliphilus was identified for the first time. The recombinant enzyme exhibited its optimal activity at pH 8.0 and 60 ℃. Additionally, the thermostability of L. secaliphilus L-ASNase was enhanced by site-directed mutagenesis after multiple sequence alignment. Ten mutants were reasonably constructed, among which the single-point mutants L24Y, S55T, and V155S showed more than 1 ℃ elevated Tm value compared to the wild-type enzyme. In addition, the half-life of mutant at 40, 50, and 55 ℃ was 376.7min, 62.1min, and 18.7min, much higher than that of wild-type enzyme. The molecular dynamic simulation showed that compared to the wild-type enzyme, the structural stability of V155S was greatly strengthened due to the lower RMSF and RMSD value as well as a decreased total energy compared to that of the wild-type enzyme. The results were positive and provided some useful information for the thermostability modification of L-ASNase.

Improvement of nutritional and bioactive properties of barley β‐glucan‐based food products using Bacillus subtilis 168 endo‐β‐1,3‐1,4‐glucanase

SummaryThe combination of β‐oligosaccharides from enzymatically hydrolysed barley β‐glucan has attracted interest recently due to its positive effects on human health. This study aimed to assess the impact of the endo‐β‐1,3‐1,4‐glucanase enzyme from Bacillus subtilis 168 on improving the nutritional and bioactive properties of barley β‐glucan. A new procedure for the isolation of β‐glucan was developed, at a lower temperature (45 °C), enabling purity from starch contamination, without affecting the yield (6 g β‐glucan from 100 g of barley flour). The endo‐β‐1,3‐1,4‐glucanase is cloned into E. coli pQE_Ek enables the high production and purification (82% yield, 1.8 mg mL−1 and 440 U mg−1) of an enzyme identical to the natural one (25.5 kDa). The enzymatic reaction showed high efficiency of β‐glucan degradation by recombinant enzyme, giving a mixture of products (of which 3‐O‐β‐cellobiosyl‐D‐glucose and 3‐O‐β‐cellotriosyl‐D‐glucose are the most abundant), the reduction of viscosity (17%) and increase in antioxidant capacities by 15.2%, 30.9% and 44.0% assessed by ABTS, DPPH and ORAC, respectively. These results indicate the possible application of endo‐β‐1,3‐1,4‐glucanase enzyme in improving the properties of barley β‐glucan used as functional foods.

Study on the Synthesis of Theaflavin-3,3′-Digallate Catalyzed by Escherichia coli Expressing Tea Tree Polyphenol Oxidase Isozymes and Its Enzymatic Solution

Polyphenol oxidase and its isoenzymes are crucial enzymes in the tea tree that catalyze the synthesis of theaflavins. In this study, tea tree polyphenol oxidase was used as the research object, and various protein sequence treatments, such as TrxA fusion tag + N-terminal truncation, were tested for prokaryotic expression through the Escherichia coli expression system. Comparative analyses were conducted on the activities of the different recombinant enzyme proteins on the substrates of tea polyphenol fractions. Additionally, the enzyme with the highest catalytic efficiency on the TFDG substrate was immobilized using polyethylene glycol to investigate the yield of its synthesis of TFDG. Our results demonstrated that after N-terminal truncation and TrxA fusion expression, CsPPO1, CsPPO2, CsPPO3, and CsPPO4 were mostly expressed in the form of inclusion bodies in the cell and exhibited varying degrees of enhancement in substrate activity. Specifically, CsPPO1 exhibited significantly increased activity in EC and ECG, CsPPO2 showed enhanced activity towards ECG and EGCG, and CsPPO2 displayed the highest activity toward TFDG substrates. Homology modeling structural analysis of the polyphenol oxidase isozymes revealed that the active centers of CsPPO1, CsPPO2, and CsPPO3 consisted of double copper ion center structures, while the conserved histidine residues surrounding the active centers formed different catalytic activity centers in different structures. Furthermore, polyethylene glycol immobilization significantly increased the activity recovery of the CsPPO2 enzyme to 74.41%. In summary, our study elucidated that tea tree polyphenol oxidase is expressed as inclusion bodies in prokaryotic expression, and the activity of the recombinant enzyme towards substrates could be enhanced through N-terminal truncation and TrxA fusion expression. Moreover, immobilization treatment of the CsPPO2 enzyme greatly improved enzyme efficiency. These findings offer an important enzymatic basis and theoretical support for the synthesis of theaflavins.

The A328 V/E (rs2887147) polymorphisms in human tryptophan hydroxylase 2 compromise enzyme activity

Human tryptophan hydroxylase 2 (hTPH2) is the rate-limiting enzyme for serotonin biosynthesis in the brain. A number of naturally-occurring single nucleotide polymorphisms (SNPs) have been reported for hTPH2. We investigated the activity and kinetic characteristics of the most common missense polymorphism rs2887147 (A328 V/E; 0.92% allelic frequency for the two different reported SNPs at the same site) using bacterially expressed hTPH2. The recombinant full-length enzyme A328E had no measurable enzyme activity, but A328V displayed decreased enzyme activity (Vmax). A328V also displayed substrate inhibition and decreased stability compared to the wild-type enzyme. By contrast, in constructs lacking the N-terminal 150 amino acid regulatory domain, the A328V substitution had no effect; that is, there was no substrate inhibition, enzyme stabilities (for wild-type and A328V) were dramatically increased, and Vmax values were not different (while the A328E variant remained inactive). These findings, in combination with molecular modeling, suggest that substitutions at A328 affect catalytic activity by altering the conformational freedom of the regulatory domain. The reduced activity and substrate inhibition resulting from these polymorphisms may ultimately reduce serotonin synthesis and contribute to behavioral perturbations, emotional stress, and eating disorders.

Characterization of a Metallic-Ions-Independent L-Arabinose Isomerase from Endophytic Bacillus amyloliquefaciens for Production of D-Tagatose as a Functional Sweetener

D-Tagatose is a low-calorie sugar substitute that has gained increased attention as a functional sweetener owing to its nutraceutical and prebiotic properties. Traditionally, D-tagatose is produced via the enzymatic conversion of L-galactose to D-tagatose by L-arabinose isomerase (L-AI). Nonetheless, the most reported L-AI enzymes are ion-dependent enzymes requiring Mn2+ and/or Co2+ as cofactors for their reactions, which limits their application due to safety and health concerns. Herein, we addressed the facile bioconversion of L-galactose to D-tagatose using a novel recombinant metallic-ions-independent L-AI derived from endophytic Bacillus amyloliquefaciens CAAI isolated from cantaloupe fruits. The ORF (1500 bp) of the L-arabinose isomerase gene (araA) was cloned and over-expressed in Escherichia coli. The recombinant enzyme (BAAI) was purified to homogeneity using Ni-NTA affinity chromatography, yielding a single distinct band with an apparent molecular mass of approximately 59 kDa as deduced from SDS-PAGE analysis. The purified enzyme showed optimum activity at pH and temperature of 7.5 and 45 °C, respectively, with obvious enzymatic activity in the presence of ethylenediaminetetraacetic acid (EDTA), indicating the metallic-ions independence from BAAI. The Km values of BAAI for D-galactose and L-arabinose were 251.6 mM and 92.8 mM, respectively. The catalytic efficiency (kcat/Km) values for D-galactose and L-arabinose were found to be 2.34 and 46.85 mM–1 min–1, respectively. The results revealed the production of 47.2 g/L D-tagatose from D-galactose (100 g/L) with 47.2% bioconversion efficiency in a metallic-ions-free reaction system that could be implemented in safe-production of food-grade low-calorie sweetener, D-tagatose.

An ACE2-Based Bimodular Fusion Protein Enables Reorientation of Endogenous Anti-Epstein-Barr Virus Antibodies Toward SARS-CoV-2 Spike.

The use of soluble recombinant angiotensin-converting enzyme 2 (rACE2) as a decoy capable of blocking SARS-CoV-2 entry into cells has been envisaged as a therapeutic strategy to reduce viral loads in patients with severe COVID-19. We engineered a novel form of rACE2, fused to the Epstein-Barr virus antigen P18F3 (rACE2-P18F3), to reorient a preexisting humoral response toward Epstein-Barr virus against SARS-CoV-2 particles. Recombinant ACE2-P18F3 was able to bind to the SARS-CoV-2 spike protein, neutralize viral entry into cells, and promote the phagocytosis of spheres coated with different spike variants by monocytic cells. The results position rACE2-P18F3 as a promising therapeutic candidate to universally block coronavirus cell entry and clear viral particles.

Purification and Activity of the Second Recombinant Enzyme for Biodegrading Linearized Microcystins by Sphingopyxis sp. USTB-05.

Hepatotoxic microcystins (MCs) are produced and released by the harmful bloom-forming cyanobacteria, which severely threaten drinking water safety and human health due to their high toxicity, widespread distribution, and structural stability. The linearized microcystinase (MlrB) further hydrolyses the poisonous linearized MCs produced by the microcystinase-catalysed MCs to form tetrapeptides. Here, the purification and activity of MlrB were investigated. The results showed that the linearized products generated by 12.5 mg/L MC-LR and MC-RR were removed by purified recombinant MlrB at a protein concentration of 1 mg/L within 30 min. The high catalytic activity of MlrB can be obtained via heterologous expression and affinity purification, which lays the foundation for further studies on the properties and mechanism of MCs biodegradation enzymes.

In vitro inhibition of human cytochrome P450 enzymes by licoisoflavone B from Glycyrrhiza uralensis Fisch. ex DC.

Glycyrrhiza uralensis Fisch. ex DC, one of the 3 pharmacopeial species of licorice and widely used in dietary supplements, can inhibit certain cytochrome P450 (CYP) enzymes. Thereby, G. uralensis preparations have the potential to cause pharmacokinetic drug interactions when consumed along with prescription medicines. One compound (1.34 mg dry weight) responsible for inhibiting CYP2B6, CYP2C8, and CYP2C9 was isolated using bioactivity-guided fractionation from 250 g dried roots, stolons, and rhizomes. The enzyme kinetics and mechanisms of inhibition were determined using human liver microsomes, recombinant enzymes, and UHPLC-MS/MS-based assays. Identified as licoisoflavone B, this compound displayed reversible inhibition of CYP2C8 with an IC50 value of 7.4 ± 1.1 µM and reversible inhibition of CYP2C9 with an IC50 value of 4.9 ± 0.4 µM. The enzyme kinetics indicated that the mechanism of inhibition was competitive for recombinant CYP2C8, with a Ki value of 7.0 ± 0.7 μM, and mixed-type inhibition for recombinant CYP2C9, with a Ki value of 1.2 ± 0.2 μM. Licoisoflavone B moderately inhibited CYP2B6 through a combination of irreversible and reversible mechanisms with an IC50 value of 16.0 ± 3.9 µM.

Search for Inhibitors of Mycobacterium tuberculosis Transketolase in a Series of Sulfo-Substituted Compounds.

As a result of the computer screening of a library of sulfo-substituted compounds, molecules capable of binding to the active site of transketolase from Mycobacterium tuberculosis were identified. An experimental verification of the inhibitory activity of the most promising compound, STK045765, against a highly purified recombinant enzyme preparation was carried out. It was shown that the STK045765 molecule competes for the binding site of the pyrophosphate group of the thiamine diphosphate cofactor and, at a micromolar concentrations, is able to suppress the activity of mycobacterial transketolase. The discovered furansulfonate scaffold may serve as the basis for the creation of anti-tuberculosis drugs.