Inhibition of Shikimate Kinase from Methicillin-Resistant Staphylococcus aureus by Benzimidazole Derivatives. Kinetic, Computational, Toxicological, and Biological Activity Studies.

Antimicrobial resistance (AMR) is one of the biggest threats in modern times. It was estimated that in 2019, 1.27 million deaths occurred around the globe due to AMR. Methicillin-resistant Staphylococcus aureus (MRSA) strains, a pathogen considered of high priority by the World Health Organization, have proven to be resistant to most of the actual antimicrobial treatments. Therefore, new treatments are required to be able to manage this increasing threat. Under this perspective, an important metabolic pathway for MRSA survival, and absent in mammals, is the shikimate pathway, which is involved in the biosynthesis of chorismate, an intermediate for the synthesis of aromatic amino acids, folates, and ubiquinone. Therefore, the enzymes of this route have been considered good targets to design novel antibiotics. The fifth step of the route is performed by shikimate kinase (SK). In this study, an in-house chemical library of 170 benzimidazole derivatives was screened against MRSA shikimate kinase (SaSK). This effort led to the identification of the first SaSK inhibitors, and the two inhibitors with the greatest inhibition activity (C1 and C2) were characterized. Kinetic studies showed that both compounds were competitive inhibitors with respect to ATP and non-competitive for shikimate. Structural analysis through molecular docking and molecular dynamics simulations indicated that both inhibitors interacted with ARG113, an important residue involved in ATP binding, and formed stable complexes during the simulation period. Biological activity evaluation showed that both compounds were able to inhibit the growth of a MRSA strain. Mitochondrial assays showed that both compounds modify the activity of electron transport chain complexes. Finally, ADMETox predictions suggested that, in general, C1 and C2 can be considered as potential drug candidates. Therefore, the benzimidazole derivatives reported here are the first SaSK inhibitors, representing a promising scaffold and a guide to design new drugs against MRSA.

Design, synthesis, kinetic, molecular dynamics, and hypoglycemic effect characterization of new and potential selective benzimidazole derivatives as Protein Tyrosine Phosphatase 1B inhibitors.

Protein-tyrosine phosphatase 1B (PTP1B) is a negative regulator of insulin signaling pathway and has been validated as a therapeutic target for type 2 diabetes. A wide variety of scaffolds have been included in the structure of PTP1B inhibitors, one of them is the benzimidazole nucleus. Here, we report the design and synthesis of a new series of di- and tri- substituted benzimidazole derivatives including their kinetic and structural characterization as PTP1B inhibitors and hypoglycemic activity. Results show that compounds 43, 44, 45, and 46 are complete mixed type inhibitors with a Ki of 12.6 µM for the most potent (46). SAR type analysis indicates that a chloro substituent at position 6(5), a ß-naphthyloxy at position 5(6), and a p-benzoic acid attached to the linker 2-thioacetamido at position 2 of the benzimidazole nucleus, was the best combination for PTP1B inhibition and hypoglycemic activity. In addition, molecular dynamics studies suggest that these compounds could be potential selective inhibitors from other PTPs such as its closest homologous TCPTP, SHP-1, SHP-2 and CDC25B. Therefore, the compounds reported here are good hits that provide structural, kinetic, and biological information that can be used to develop novel and selective PTP1B inhibitors based on benzimidazole scaffold.

Benzimidazole Derivatives as New and Selective Inhibitors of Arginase from Leishmania mexicana with Biological Activity against Promastigotes and Amastigotes.

Leishmaniasis is a disease caused by parasites of the Leishmania genus that affects 98 countries worldwide, 2 million of new cases occur each year and more than 350 million people are at risk. The use of the actual treatments is limited due to toxicity concerns and the apparition of resistance strains. Therefore, there is an urgent necessity to find new drugs for the treatment of this disease. In this context, enzymes from the polyamine biosynthesis pathway, such as arginase, have been considered a good target. In the present work, a chemical library of benzimidazole derivatives was studied performing computational, enzyme kinetics, biological activity, and cytotoxic effect characterization, as well as in silico ADME-Tox predictions, to find new inhibitors for arginase from Leishmania mexicana (LmARG). The results show that the two most potent inhibitors (compounds 1 and 2) have an I50 values of 52 µM and 82 µM, respectively. Moreover, assays with human arginase 1 (HsARG) show that both compounds are selective for LmARG. According to molecular dynamics simulation studies these inhibitors interact with important residues for enzyme catalysis. Biological activity assays demonstrate that both compounds have activity against promastigote and amastigote, and low cytotoxic effect in murine macrophages. Finally, in silico prediction of their ADME-Tox properties suggest that these inhibitors support the characteristics to be considered drug candidates. Altogether, the results reported in our study suggest that the benzimidazole derivatives are an excellent starting point for design new drugs against leishmanisis.

Finding the First Potential Inhibitors of Shikimate Kinase from Methicillin Resistant Staphylococcus aureus through Computer-Assisted Drug Design.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important threat as it causes serious hospital and community acquired infections with deathly outcomes oftentimes, therefore, development of new treatments against this bacterium is a priority. Shikimate kinase, an enzyme in the shikimate pathway, is considered a good target for developing antimicrobial drugs; this is given because of its pathway, which is essential in bacteria whereas it is absent in mammals. In this work, a computer-assisted drug design strategy was used to report the first potentials inhibitors for Shikimate kinase from methicillin-resistant Staphylococcus aureus (SaSK), employing approximately 5 million compounds from ZINC15 database. Diverse filtering criteria, related to druglike characteristics and virtual docking screening in the shikimate binding site, were performed to select structurally diverse potential inhibitors from SaSK. Molecular dynamics simulations were performed to elucidate the dynamic behavior of each SaSK-ligand complex. The potential inhibitors formed important interactions with residues that are crucial for enzyme catalysis, such as Asp37, Arg61, Gly82, and Arg138. Therefore, the compounds reported provide valuable information and can be seen as the first step toward developing SaSK inhibitors in the search of new drugs against MRSA.

Leishmania mexicana Trypanothione Reductase Inhibitors: Computational and Biological Studies.

Leishmanicidal drugs have many side effects, and drug resistance to all of them has been documented. Therefore, the development of new drugs and the identification of novel therapeutic targets are urgently needed. Leishmania mexicana trypanothione reductase (LmTR), a NADPH-dependent flavoprotein oxidoreductase important to thiol metabolism, is essential for parasite viability. Its absence in the mammalian host makes this enzyme an attractive target for the development of new anti-Leishmania drugs. Herein, a tridimensional model of LmTR was constructed and the molecular docking of 20 molecules from a ZINC database was performed. Five compounds (ZINC04684558, ZINC09642432, ZINC12151998, ZINC14970552, and ZINC11841871) were selected (docking scores -10.27 kcal/mol to -5.29 kcal/mol and structurally different) and evaluated against recombinant LmTR (rLmTR) and L. mexicana promastigote. Additionally, molecular dynamics simulation of LmTR-selected compound complexes was achieved. The five selected compounds inhibited rLmTR activity in the range of 32.9% to 40.1%. The binding of selected compounds to LmTR involving different hydrogen bonds with distinct residues of the molecule monomers A and B is described. Compound ZINC12151998 (docking score -10.27 kcal/mol) inhibited 32.9% the enzyme activity (100 µM) and showed the highest leishmanicidal activity (IC50 = 58 µM) of all the selected compounds. It was more active than glucantime, and although its half-maximal cytotoxicity concentration (CC50 = 53 µM) was higher than that of the other four compounds, it was less cytotoxic than amphotericin B. Therefore, compound ZINC12151998 provides a promising starting point for a hit-to-lead process in our search for new anti-Leishmania drugs that are more potent and less cytotoxic.

Novel Mixed-Type Inhibitors of Protein Tyrosine Phosphatase 1B. Kinetic and Computational Studies.

The Atlas of Diabetes reports 415 million diabetics in the world, a number that has surpassed in half the expected time the twenty year projection. Type 2 diabetes is the most frequent form of the disease; it is characterized by a defect in the secretion of insulin and a resistance in its target organs. In the search for new antidiabetic drugs, one of the principal strategies consists in promoting the action of insulin. In this sense, attention has been centered in the protein tyrosine phosphatase 1B (PTP1B), a protein whose overexpression or increase of its activity has been related in many studies with insulin resistance. In the present work, a chemical library of 250 compounds was evaluated to determine their inhibition capability on the protein PTP1B. Ten molecules inhibited over the 50% of the activity of the PTP1B, the three most potent molecules were selected for its characterization, reporting Ki values of 5.2, 4.2 and 41.3 µM, for compounds 1, 2, and 3, respectively. Docking and molecular dynamics studies revealed that the three inhibitors made interactions with residues at the secondary binding site to phosphate, exclusive for PTP1B. The data reported here support these compounds as hits for the design more potent and selective inhibitors against PTP1B in the search of new antidiabetic treatment.

Species-Specific Inactivation of Triosephosphate Isomerase from Trypanosoma brucei: Kinetic and Molecular Dynamics Studies.

Human African Trypanosomiasis (HAT), a disease that provokes 2184 new cases a year in Sub-Saharan Africa, is caused by Trypanosoma brucei. Current treatments are limited, highly toxic, and parasite strains resistant to them are emerging. Therefore, there is an urgency to find new drugs against HAT. In this context, T. brucei depends on glycolysis as the unique source for ATP supply; therefore, the enzyme triosephosphate isomerase (TIM) is an attractive target for drug design. In the present work, three new benzimidazole derivatives were found as TbTIM inactivators (compounds 1, 2 and 3) with an I50 value of 84, 82 and 73 µM, respectively. Kinetic analyses indicated that the three molecules were selective when tested against human TIM (HsTIM) activity. Additionally, to study their binding mode in TbTIM, we performed a 100 ns molecular dynamics simulation of TbTIM-inactivator complexes. Simulations showed that the binding of compounds disturbs the structure of the protein, affecting the conformations of important domains such as loop 6 and loop 8. In addition, the physicochemical and drug-like parameters showed by the three compounds suggest a good oral absorption. In conclusion, these molecules will serve as a guide to design more potent inactivators that could be used to obtain new drugs against HAT.

Synthesis and trypanocidal activity of novel benzimidazole derivatives.

The present work reports the synthesis and biological activity of a series of 14 benzimidazole derivatives designed to act on the enzyme triosephosphate isomerase of Trypanosoma cruzi (TcTIM). This enzyme is involved in the metabolism of glucose, the only source of energy for the parasite. In this study, we found four compounds that inhibit TcTIM moderately and lack inhibitory activity against human TIM (HsTIM). In vitro studies against T. cruzi epimastigotes showed two compounds that were more active than the reference drug nifurtimox, and these presented a low cytotoxic effect in mouse macrophages (J744 cell line).

Arsenic exposure and risk of preeclampsia in a Mexican mestizo population.

BACKGROUND: Exposure to arsenic in drinking water has been associated with various complications of pregnancy including fetal loss, low birth weight, anemia, gestational diabetes and spontaneous abortion. However, to date, there are no studies evaluating its possible association with preeclampsia. METHODS: This case-control study involved 104 preeclamptic and 202 healthy pregnant women. The concentrations of arsenic in drinking water and urine were measured using a Microwave Plasma-Atomic Emission Spectrometer. RESULTS: We found relatively low levels of arsenic in household tap water (range of 2.48-76.02 µg/L) and in the urine of the participants (7.1 µg/L vs 6.78 µg/L in cases and controls, respectively). CONCLUSIONS: The analysis between groups showed for the first time that at these lower levels of exposure there is no association with preeclampsia.

Discovery of Entamoeba histolytica hexokinase 1 inhibitors through homology modeling and virtual screening.

Entamoeba histolytica, the parasite which causes amebiasis is responsible for 110,000 deaths a year. Entamoeba histolytica depends on glycolysis to obtain ATP for cellular work. According to metabolic flux studies, hexokinase exerts the highest flux control of this metabolic pathway; therefore, it is an excellent target in the search of new antiamebic drugs. To this end, a tridimensional model of E. histolytica hexokinase 1 (EhHK1) was constructed and validated by homology modeling. After virtual screening of 14,400 small molecules, the 100 with the best docking scores were selected, purchased and assessed in their inhibitory capacity. The results showed that three molecules (compounds 2921, 11275 and 2755) inhibited EhHK1 with an I50 of 48, 91 and 96 µM, respectively. Thus, we found the first inhibitors of EhHK1 that can be used in the search of new chemotherapeutic agents against amebiasis.

Polymorphisms in DNA repair genes (APEX1, XPD, XRCC1 and XRCC3) and risk of preeclampsia in a Mexican mestizo population.

Variations in genes involved in DNA repair systems have been proposed as risk factors for the development of preeclampsia (PE). We conducted a case-control study to investigate the association of Human apurinic/apyrimidinic (AP) endonuclease (APEX1) Asp148Glu (rs1130409), Xeroderma Pigmentosum group D (XPD) Lys751Gln (rs13181), X-ray repair cross-complementing group 1 (XRCC) Arg399Gln (rs25487) and X-ray repair cross-complementing group 3 (XRCC3) Thr241Met (rs861539) polymorphisms with PE in a Mexican population. Samples of 202 cases and 350 controls were genotyped using RTPCR. Association analyses based on a χ2 test and binary logistic regression were performed to determine the odds ratio (OR) and a 95% confidence interval (95% CI) for each polymorphism. The allelic frequencies of APEX1 Asp148Glu polymorphism showed statistical significant differences between preeclamptic and normal women (p = 0.036). Although neither of the polymorphisms proved to be a risk factor for the disease, the APEX1 Asp148Glu polymorphism showed a tendency of association (OR: 1.74, 95% CI = 0.96-3.14) and a significant trend (p for trend = 0.048). A subgroup analyses revealed differences in the allelic frequencies of APEX1 Asp148Glu polymorphism between women with mild preeclampsia and severe preeclampsia (p = 0.035). In conclusion, our results reveal no association between XPD Lys751Gln, XRCC Arg399Gln and XRCC3 Thr241Met polymorphisms and the risk of PE in a Mexican mestizo population; however, the results in the APEX1 Asp148Glu polymorphism suggest the need for future studies using a larger sample size.

Inhibition and biochemical characterization of methicillin-resistant Staphylococcus aureus shikimate dehydrogenase: an in silico and kinetic study.

Methicillin-resistant Staphylococcus auerus (MRSA) strains are having a major impact worldwide, and due to their resistance to all ß-lactams, an urgent need for new drugs is emerging. In this regard, the shikimate pathway is considered to be one of the metabolic features of bacteria and is absent in humans. Therefore enzymes involved in this route, such as shikimate dehydrogenase (SDH), are considered excellent targets for discovery of novel antibacterial drugs. In this study, the SDH from MRSA (SaSDH) was characterized. The results showed that the enzyme is a monomer with a molecular weight of 29 kDa, an optimum temperature of 65 °C, and a maximal pH range of 9-11 for its activity. Kinetic studies revealed that SDH showed Michaelis-Menten kinetics toward both substrates (shikimate and NADP+). Initial velocity analysis suggested that SaSDH catalysis followed a sequential random mechanism. Additionally, a tridimensional model of SaSDH was obtained by homology modeling and validated. Through virtual screening three inhibitors of SaSDH were found (compounds 238, 766 and 894) and their inhibition constants and mechanism were obtained. Flexible docking studies revealed that these molecules make interactions with catalytic residues. The data of this study could serve as starting point in the search of new chemotherapeutic agents against MRSA.

Genetic variation in oxidative stress and DNA repair genes in a Mexican population.

BACKGROUND: Oxidative stress has been associated with several complex diseases. Effects generated as a result of oxidative stress may be modulated by various genes. Variation in these genes, particularly when located within coding or regulating regions, may be the primary cause of this modulation. The aim of this work was to determine the allelic and genotypic frequencies of CAT C-262T, SOD3 Ala58Thr, APEX1 Asp148Glu, XPD Lys751Gln and XRCC3 Thr241Met genetic markers in a northern Mexican population. SUBJECTS AND METHODS: This study analysed 250 unrelated individuals by RT-PCR. RESULTS: A high allele mutant frequency was found in SOD3 Ala58Thr and APEX1 Asp148Glu genetic markers (0.395 and 0.38, respectively). A correspondence analysis showed that northern Mexicans are close to European populations. A linkage disequilibrium test between XPD Lys751Gln and CAT C-262T and XPD Lys751Gln and SOD3 Ala58Thr genetic markers was significant (p = 0.000). CONCLUSION: The genetic markers described in this work will be a valuable resource for future functional studies in the northern Mexican population to explore comprehensively their role in the aetiology of human diseases. Furthermore, it will be necessary to replicate these studies in other regions of Mexico due to differences between Mexican sub-populations.

Discovery of Potential Noncovalent Inhibitors of Dehydroquinate Dehydratase from Methicillin-Resistant Staphylococcus aureus through Computational-Driven Drug Design.

Bacteria resistance to antibiotics is a concerning global health problem; in this context, methicillin-resistant Staphylococcus aureus (MRSA) is considered as a high priority by the World Health Organization. Furthermore, patients with a positive result for COVID-19 received early antibiotic treatment, a fact that potentially encourages the increase in antibiotic resistance. Therefore, there is an urgency to develop new drugs with molecular mechanisms different from those of the actual treatments. In this context, enzymes from the shikimate pathway, a route absent in humans, such as dehydroquinate dehydratase (DHQD), are considered good targets. In this work, a computer-aided drug design strategy, which involved exhaustive virtual screening and molecular dynamics simulations with MM-PBSA analysis, as well as an in silico ADMETox characterization, was performed to find potential noncovalent inhibitors of DHQD from MRSA (SaDHQD). After filtering the 997 million compounds from the ZINC database, 6700 compounds were submitted to an exhaustive virtual screening protocol. From these data, four molecules were selected and characterized (ZINC000005753647 (1), ZINC000001720488 (2), ZINC000082049768 (3), and ZINC000644149506 (4)). The results indicate that the four potential inhibitors interacted with residues important for substrate binding and catalysis, with an estimated binding free energy like that of the enzyme's substrate. Their ADMETox-predicted properties suggest that all of them support the structural characteristics to be considered good candidates. Therefore, the four compounds reported here are excellent option to be considered for future in vitro studies to design new SaDHQD noncovalent inhibitors and contribute to the search for new drugs against MRSA.

In Silico Characterization of the Physicochemical and Biological Properties of the Pink (Pleurotus djamor var. salmoneostramineus) Oyster Mushroom Chromoprotein.

Cap color is an important commercial trait for oyster mushrooms. Various pigment constituents determine a diverse color. However, the pigments of oyster mushrooms are still ambiguous. The pink oyster mushroom (Pleurotus salmoneostramineus or Pleurotus djamor) chromoprotein is one of the few proteins belonging to this fungus that has a record of its sequence of amino acid residues. However, even though there are studies about this chromoprotein isolation, purification, and crystallization, the current information focused on its 3-dimensional model and the cofactor and prosthetic group (3H-indol-3-one) binding sites is unreliable and fragmented. Therefore, in this study, using free online servers such as Prot pi, GalaxyWEB, MIB, and CB-Dock2, a structural analysis and the prediction of its physicochemical and biological properties were conducted, to understand the possible function of this chromoprotein. The obtained results showed that this molecule is a protein with a molecular weight of 23 712.5 Da, an isoelectric point of 7.505, with oligomerization capacity in a dimer and glycation in the Ser6 residue. In addition, the participation of the residues Leu5, Leu8, Lys211, Ala214, and Gln215 in the binding of the prosthetic group to the protein was highlighted; as well as Ser6 and Pro7 are important residues for the interaction of the Mg2+ ion and eumelanin. Likewise, morphological changes based on different culture conditions (light/dark) showed that this protein is constitutive expressed and independent of blue light. The findings in this study demonstrate that pink chromoprotein is a melanosomal protein, and it possibly has a critical role in melanogenesis and the melanin polymerization. However, more experimental studies are needed to predict a possible mechanism of action and type of enzymatic activity.

Characterization of Moringa oleifera Seed Oil for the Development of a Biopackage Applied to Maintain the Quality of Turkey Ham.

Moringa oleifera has a high level of active chemicals that are useful in the food industry, and they have antibacterial and food preservation properties. The characterization of M. oleifera seed oil (MOS) may vary due to agronomic and environmental factors. Therefore, it was necessary to know the composition of lipids present in our oil extracted under pressing at 180 °C and thus determine if it is suitable to produce a biopackaging. Within the characterization of the oil, it was obtained that MOS presented high-quality fatty acids (71% oleic acid) with low values of acidity (0.71 mg KOH/g) and peroxide (1.74 meq O2/kg). Furthermore, MOS was not very sensitive to lipoperoxidation by tert-butyl hydroperoxide (tBuOOH) and its phenolic components, oleic acid and tocopherols, allowed MOS to present a recovery of 70% after 30 min of treatment. Subsequently, a biopackaging was developed using a multiple emulsion containing corn starch/carboxymethylcellulose/glycerol/MOS, which presented good mechanical properties (strength and flexibility), transparency, and a barrier that prevents the transfer of UV light by 30% and UV-C by 98%, as well as a flux with the atmosphere of 5.12 × 10-8 g/ m.s. Pa that prevents moisture loss and protects the turkey ham from O2. Hence, the turkey ham suffered less weight loss and less hardness due to its preservation in the biopackaging.

Computational Methods in Cooperation with Experimental Approaches to Design Protein Tyrosine Phosphatase 1B Inhibitors in Type 2 Diabetes Drug Design: A Review of the Achievements of This Century.

Protein tyrosine phosphatase 1B (PTP1B) dephosphorylates phosphotyrosine residues and is an important regulator of several signaling pathways, such as insulin, leptin, and the ErbB signaling network, among others. Therefore, this enzyme is considered an attractive target to design new drugs against type 2 diabetes, obesity, and cancer. To date, a wide variety of PTP1B inhibitors that have been developed by experimental and computational approaches. In this review, we summarize the achievements with respect to PTP1B inhibitors discovered by applying computer-assisted drug design methodologies (virtual screening, molecular docking, pharmacophore modeling, and quantitative structure-activity relationships (QSAR)) as the principal strategy, in cooperation with experimental approaches, covering articles published from the beginning of the century until the time this review was submitted, with a focus on studies conducted with the aim of discovering new drugs against type 2 diabetes. This review encourages the use of computational techniques and includes helpful information that increases the knowledge generated to date about PTP1B inhibition, with a positive impact on the route toward obtaining a new drug against type 2 diabetes with PTP1B as a molecular target.

Cordyceps militaris Inhibited Angiotensin-Converting Enzyme through Molecular Interaction between Cordycepin and ACE C-Domain.

One of the most important therapeutic modalities for the management of hypertension is the inhibition of the angiotensin-converting enzyme (ACE). Cordyceps militaris has received substantial attention because to its therapeutic potential and biological value. To gather information about the antihypertensive properties of C. militaris, the ACE inhibitory activity was evaluated. An ethanolic extract of the fruiting body of C. militaris was obtained, and the extract was separated by UHPLC method with a fluorescence detector for the quantification of cordycepin and adenosine. The ethanolic extract had a considerably higher cordycepin level. Additionally, an in vitro kinetic analysis was carried out to find out how much C. militaris extract inhibited ACE. This extract exhibited non-competitive inhibition on ACE. The Ki value of the C. militaris extract against ACE was found to be 8.7 µg/mL. To the best of our knowledge, this is the first report of the analysis of a protein cavity together with molecular docking carried out to comprehend the intermolecular interactions between cordycepin and the ACE C-domain, which impact the spatial conformation of the enzyme and reduce its capacity to break down the substrate. According to a molecular docking, hydrogen bonding interactions between the chemicals and the ACE S2' subsite are primarily responsible for cordycepin inhibition at the ACE C domain. All these findings suggest that C. militaris extract are a kind of natural ACE inhibitor, and cordycepin has the potential as an ACE inhibitor.

Effects of Moringa oleifera Leaf Extract on Diabetes-Induced Alterations in Paraoxonase 1 and Catalase in Rats Analyzed through Progress Kinetic and Blind Docking.

In our study, we aimed to evaluate the effects of Moringa oleifera leaves extract on rat paraoxonase 1 (rPON1) and catalase (rCAT) activities in alloxan-induced diabetic rats. Our study included three groups; group C (control, n = 5); group D (diabetic, n = 5); and group DM (M. oleifera extract-supplemented diabetic rats, n = 5). Daily oral administration of M. oleifera extract at 200 mg/kg doses produced an increase in endogenous antioxidants. Serum rPON1 (lactonase) and liver cytosol catalase activities were determined by a spectrophotometric assay using progress curve analysis. We found a decrease in the Vm value of rPON1 in diabetic rats, but dihydrocoumarin (DHC) affinity (Km) was slightly increased. The value of Vm for the DM group was found to be reduced approximately by a factor of 3 compared with those obtained for group C, whereas Km was largely changed (96 times). Catalase activity was significantly higher in the DM group. These data suggest that the activation of rPON1 and rCAT activities by M. oleifera extracts may be mediated via the effect of the specific flavonoids on the enzyme structure. In addition, through molecular blind docking analysis, rPON1 was found to have two binding sites for flavonoids. In contrast, flavonoids bound at four sites in rCAT. In conclusion, the data suggest that compounds from M. oleifera leaves extract were able to influence the catalytic activities of both enzymes to compensate for the changes provoked by diabetes in rats.

Engineering and directed evolution of a Ca2+ binding site A-deficient AprE mutant reveal an essential contribution of the loop Leu75-Leu82 to enzyme activity.

An aprE mutant from B. subtilis 168 lacking the connecting loop Leu(75)-Leu(82) which is predicted to encode a Ca(2+) binding site was constructed. Expression of the mutant gene (aprEDeltaLeu(75)-Leu(82)) produced B. subtilis colonies lacking protease activity. Intrinsic fluorescence analysis revealed spectral differences between wild-type AprE and AprEDeltaL(75)-L(82). An AprEDeltaL(75)-L(82) variant with reestablished enzyme activity was selected by directed evolution. The novel mutations Thr(66)Met/Gly(102)Asp located in positions which are predicted to be important for catalytic activity were identified in this variant. Although these mutations restored hydrolysis, they had no effect with respect to thermal inactivation of AprEDeltaL(75)-L(82) T(66)M G(102)D. These results support the proposal that in addition to function as a calcium binding site, the loop that connects beta-sheet e3 with alpha-helix c plays a structural role on enzyme activity of AprE from B. subtilis 168.