Rita Pacheco

Substitutions of Thr-103-Ile and Trp-138-Gly in amidase from Pseudomonas aeruginosa are responsible for altered kinetic properties and enzyme instability

Pseudomonas aeruginosa Ph1 is a mutant strain derived from strain AI3. The strain AI3 is able to use acetanilide as a carbon source through a mutation (T103I) in the amiE gene that encodes an aliphatic amidase (EC 3.5.1.4). The mutations in the amiE gene have been identified (Thr103Ile and Trp138Gly) by direct sequencing of PCR-amplified mutant gene from strain Ph1 and confirmed by sequencing the cloned PCR-amplified gene. Site-directed mutagenesis was used to alter the wild-type amidase gene at position 138 for Gly. The wild-type and mutant amidase genes (W138G, T103I-W138G, and T103I) were cloned into an expression vector and these enzymes were purified by affinity chromatography on epoxy-activated Sepharose 6B-acetamide/phenylacetamide followed by gel filtration chromatography. Altered amidases revealed several differences in kinetic properties, namely, in substrate specificity, sensitivity to urea, optimum pH, and enzyme stability, compared with the wild-type enzyme. The W138G enzyme acted on acetamide, acrylamide, phenylacetamide, and p-nitrophenylacetamide, whereas the double mutant (W138G and T103I) amidase acted only on p-nitrophenylacetamide and phenylacetamide. On the other hand, the T103I enzyme acted on p-nitroacetanilide and acetamide. The heat stability of altered enzymes revealed that they were less thermostable than the wild-type enzyme, as the mutant (W138G and W138G-T103I) enzymes exhibited t1/2 values of 7.0 and 1.5 min at 55°C, respectively. The double substitution T103I and W138G on the amidase molecule was responsible for increased instabiliby due to a conformational change in the enzyme molecule as detected by monoclonal antibodies. This conformational change in altered amidase did not alter its Mr value and monoclonal antibodies reacted differently with the active and inactive T103I-W138G amidase.

Amidase encapsulated in TTAB reversed micelles for the study of transamidation reactions

Amidase, an amide hydrolase enzyme (E.C.3.5.1.4) with acyl transferase activity, was encapsulated in a reversed micellar system composed of the cationic surfactant tetradecyltrimethyl ammonium bromide (TTAB) in heptane/octanol (80/20%) and phosphate buffer at w0 11. The reaction used to study the effect of the reversed micellar system on the enzyme behaviour was a transamidation reaction. The effect of surfactant concentration, buffer molarity and pH on the enzyme kinetics was evaluated. Both initial velocities and product yield were measured. The results indicated that a high initial velocity of hydroxamic acid synthesis and also the highest yield (98%) were obtained using the lowest pH value. The effect of TTAB concentration was dependent on the buffer molarity used. The effect of buffer molarity on reversed micelle dimensions was analysed by light scattering. These results showed that the buffer molarity had a strong influence on the reversed micelle radius that correlated with enzyme activity.

Antiacetylcholinesterase activity and docking studies with chlorogenic acid, cynarin and arzanol from Helichrysum stoechas (Lamiaceae)

This work was aimed at the study of the chemical composition in phenolic compounds responsible for the high antiacetylcholinesterase activity of aqueous extracts (decoctions) from Helichrysum stoechas aerial parts. Chlorogenic acid, cynarin, and arzanol were the main components of decoctions, detected by high-performance liquid chromatography with diode-array detection and liquid chromatography-mass spectrometry/mass spectrometry. Flowers and stems/leaves extracts inhibited antiacetylcholinesterase with IC50 values of 260.7 and 654.8 μg/mL, respectively. The biological activity of these extracts was maintained after in vitro gastrointestinal digestion, indicating that the active compounds present in the extracts were not enzymatically modified by the gastrointestinal system used to simulate the digestion. Molecular docking studies with the main components were carried out in order to obtain information, at the molecular level, as to how these compounds access the enzyme’s active site. The docking study showed for the first time that chlorogenic acid, cynarin, and arzanol fit nicely in the antiacetylcholinesterase active site channel, blocking all access to the catalytic triad. This explained the high inhibitory activity determined during in vitro experiments.

Pseudomonas aeruginosa amidase: Aggregation in recombinant Escherichia coli

The effect of cultivation parameters such as temperature incubation, IPTG induction and ethanol shock on the production of Pseudomonas aeruginosa amidase (E.C.3.5.1.4) in a recombinant Escherichia coli strain in LB ampicillin culture medium was investigated. The highest yield of soluble amidase, relatively to other proteins, was obtained in the condition at 37°C using 0.40 mM IPTG to induce growth, with ethanol. Our results demonstrate the formation of insoluble aggregates containing amidase, which was biologically active, in all the tested growth conditions. Addition of ethanol at 25°C in the culture medium improved amidase yield, which quantitatively aggregated in a biological active form and exhibited in all conditions an increased specific activity relatively to the soluble form of the enzyme. Non-denaturing solubilization of the aggregated amidase was successfully achieved using L-arginine. The aggregates obtained from conditions at 37°C by FTIR analysis demonstrated a lower content of intermolecular interactions which facilitated the solubilization step applying non-denaturing conditions. The higher interactions exhibited in aggregates obtained at suboptimal conditions compromised the solubilization yield. This work provides an approach for the characterization and solubilization of novel reported biologically active aggregates of this amidase.

Bioactivities of decoctions from Plectranthus species related to their traditional use on the treatment of digestive problems and alcohol intoxication

ETHNOPHARMACOLOGICAL RELEVANCE Decoctions of Plectranthus species are traditionally ingested after large meals for treatment of food digestion and alcohol abuse. AIM OF THE STUDY This study aims at associating the digestion-related ethno-uses of Plectranthus species decoctions to molecular mechanism that might explain them: easing digestion (AChE inhibition) and treating hangover (ADH inhibition) MATERIAL AND METHODS: Decoctions from Plectranthus species were analysed for their alcohol dehydrogenase (ADH) inhibition and acetylcholinesterase (AChE) inhibition, related with alcohol metabolism and intestinal motility, respectively. Identification of the active components was carried out by LC-MS/MS and the docking studies were performed with AChE and the bioactive molecules detected. RESULTS All decoctions inhibited ADH activity. This inhibition was correlated with their rosmarinic acid (RA) content, which showed an IC50 value of 19 μg/mL, similar to the reference inhibitor CuCl2. The presence of RA also leads to most decoctions showing AChE inhibiting capacity. P. zuluensis decoction with an IC50 of 80 μg/mL presented also medioresinol, an even better inhibitor of AChE, as indicated by molecular docking studies. Furthermore, all decoctions tested showed no toxicity towards two human cell lines, and a high capacity to quench free radicals (DPPH), which also play a helpful in the digestive process, related with their RA content. CONCLUSIONS All activities presented by the RA-rich Plectranthus decoctions support their use in treating digestion disorders and P. barbatus could explain its use also for alleviating hangover symptoms. Medioresinol, which is present in P. zuluensis, exhibited a significant AChE inhibition and may provide, in the future, a new lead for bioactive compounds.

Proactive response to tackle the threat of emerging drugs: Synthesis and toxicity evaluation of new cathinones

The emergence of potentially dangerous new psychoactive substances (NPS) imposes enormous challenges on forensic laboratories regarding their rapid and unambiguous identification. Access to comprehensive databases is essential for a quick characterization of these substances, allowing them to be categorized according to national and international legislations. In this work, it is reported the synthesis and structural characterization by NMR and MS of a library encompassing 21 cathinones, 4 of which are not yet reported in the literature, but with structural characteristics that make them a target for clandestine laboratories. This in-house library will be an important tool accessible to forensic laboratories, for the quick identification of seized NPS. The in vitro cytotoxicity of all cathinones was assessed in HepG2 cells, to have a preliminary but effective indication of their human hepatotoxicity potential. The two new cathinones DMB (8) and DMP (9) were the more cytotoxic, followed by the already seized mephedrone (2), 3,4-DMMC (3), 4-MDMC (7), NEB (12) with EC50 values ranging from 0.81mM for (3) to 1.28mM for (2). Results suggest an increase of cytotoxicity with the increase of the chain length of the acyl moiety and with the substitution (with one or two methyl groups) in the aromatic ring. The nature of the amine moiety seems to play only a minor role in the cytotoxic effect. Molecular dynamics simulations were performed to evaluate the molecular details related with the observed cytotoxicities. Although these studies indicated that cathinones are able to cross lipid bilayers with relative ease, when in their neutral forms, it was observed only a partial correlation between lipophilicity and cytotoxicity, indicating that membrane trafficking may not be the only key factor influencing the bioactivity of these compounds. This work is a valuable contribution to the forensic science field since a quick identification of novel cathinones is urgent to match their rapid increase in the market.

Correction to: Antiacetylcholinesterase activity and docking studies with chlorogenic acid, cynarin and arzanol from Helichrysum stoechas (Asteraceae)

The original version of this article unfortunately contained an error in the article title. There is a incorrect term Lamiaceae inadvertently appeared in the title, instead it should be Asteraceae.

Serum Albumin Modulates the Bioactivity of Rosmarinic Acid

Rosmarinic acid (RA) is a phenolic compound with biological activity. The objective of the present study was to investigate whether this compound kept its biological activity in the presence of proteins. For this purpose, bovine serum albumin (BSA) was used as a model protein, and the capacity of the RA to inhibit acetylcholinesterase (AChE) and affect antioxidant activity was evaluated in the absence and presence of BSA. A mixture of phenolic compounds containing RA, obtained from a medicinal plant was added to this study. The AChE inhibitory activity of RA was reduced by ∼57% in the presence of BSA, while the antioxidant activity increased. These results lead to the investigation of the effect of RA on the BSA structure using Fourier transform infrared spectroscopy (FTIR). At 37°C and higher temperatures, RA caused a decrease in the temperature modifications on the protein structure. Furthermore, FTIR and native-gel analysis revealed that protein aggregation/precipitation, induced by temperature, was reduced in the presence of RA. The novelty of the present work resides in the study of the enzyme inhibitory activity and antioxidant capacity of polyphenols, such as RA, in the presence of a protein. The findings highlight the need to consider the presence of proteins when assessing biological activities of polyphenols in vitro and that enzyme inhibitory activity may be decreased, while the antioxidant capacity remains or even increases.

Substrate interaction with recombinant amidase from Pseudomonas aeruginosa during biocatalysis

The interaction of a variety of substrates with Pseudomonas aeruginosa native amidase (E.C. 3.5.1.4), overproduced in an Escherichia coli strain, was investigated using difference FTIR spectroscopy. The amides used as substrates showed an increase in hydrogen bonding upon association in multimers, which was not seen with esters. Evidence for an overall reduction or weakening of hydrogen bonding while amide and ester substrates are interacting with the enzyme is presented. The results describe a spectroscopic approach for analysis of substrate–amidase interaction and in situ monitoring of the hydrolysis and transferase reaction when amides or esters are used as substrates.