Insights into tRNA-Dependent Amidotransferase Evolution and Catalysis from the Structure of the Aquifex aeolicus Enzyme

Many bacteria form Gln-tRNA^Gln and Asn-tRNA^Asn by conversion of the misacylated Glu-tRNA^Gln and Asp-tRNA^Asn species catalyzed by the GatCAB amidotransferase in the presence of ATP and an amide donor (glutamine or asparagine). Here, we report the crystal structures of GatCAB from the hyperthermophilic bacterium Aquifex aeolicus, complexed with glutamine, asparagine, aspartate, ADP, or ATP. In contrast to the Staphylococcus aureus GatCAB, the A. aeolicus enzyme formed acyl-enzyme intermediates with either glutamine or asparagine, in line with the equally facile use by the amidotransferase of these amino acids as amide donors in the transamidation reaction.A water-filled ammonia channel is open throughout the length of the A. aeolicus GatCAB from the GatA active site to the synthetase catalytic pocket in the B-subunit. A non-catalytic Zn²⁺ site in the A. aeolicus GatB stabilizes subunit contacts and the ammonia channel. Judged from sequence conservation in the known GatCAB sequences, the Zn²⁺ binding motif was likely present in the primordial GatB/E, but became lost in certain lineages (e.g., S. aureus GatB). Two divalent metal binding sites, one permanent and the other transient, are present in the catalytic pocket of the A. aeolicus GatB. The two sites enable GatCAB to first phosphorylate the misacylated tRNA substrate and then amidate the activated intermediate to form the cognate products, Gln-tRNA^Gln or Asn-tRNA^Asn.     

Structure and characterization of amidase from Rhodococcus sp. N-771: Insight into the molecular mechanism of substrate recognition

In this study, we have structurally characterized the amidase of a nitrile-degrading bacterium, Rhodococcus sp. N-771 (RhAmidase). RhAmidase belongs to amidase signature (AS) family, a group of amidase families, and is responsible for the degradation of amides produced from nitriles by nitrile hydratase. Recombinant RhAmidase exists as a dimer of about 107 kDa. RhAmidase can hydrolyze acetamide, propionamide, acrylamide and benzamide with kcat/Km values of 1.14 ± 0.23 mM^− 1s^− 1, 4.54 ± 0.09 mM^− 1s^− 1, 0.087 ± 0.02 mM^− 1s^− 1 and 153.5 ± 7.1 mM^− 1s^− 1, respectively. The crystal structures of RhAmidase and its inactive mutant complex with benzamide (S195A/benzamide) were determined at resolutions of 2.17 Å and 2.32 Å, respectively. RhAmidase has three domains: an N-terminal α-helical domain, a small domain and a large domain. The N-terminal α-helical domain is not found in other AS family enzymes. This domain is involved in the formation of the dimer structure and, together with the small domain, forms a narrow substrate-binding tunnel. The large domain showed high structural similarities to those of other AS family enzymes. The Ser-cis Ser-Lys catalytic triad is located in the large domain. But the substrate-binding pocket of RhAmidase is relatively narrow, due to the presence of the helix α13 in the small domain. The hydrophobic residues from the small domain are involved in recognizing the substrate. The small domain likely participates in substrate recognition and is related to the difference of substrate specificities among the AS family amidases.     

A Comparative Analysis of the ‘Green’ Techniques Applied for Polyphenols Extraction from Bioresources

From all the valuable biomass extractives, polyphenols are a widespread group of secondary metabolites found in all plants, representing the most desirable phytochemicals due to their potential to be used as additives in food industry, cosmetics, medicine, and others fields. At present, there is an increased interest to recover them from plant of spontaneous flora, cultivated plant, and wastes resulted in agricultural and food industry. That is why many efforts have been made to provide a highly sensitive, efficiently, and eco‐friendly methods, for the extraction of polyphenols, according to the green chemistry and sustainable development concepts. Many extraction procedures are known with advantages and disadvantages. From these reasons, the aim of this article is to provide a comparative analysis regarding technical and economical aspects related to the most innovative extraction techniques studied in the last time: microwave‐assisted extraction (MAE), supercritical fluid extraction (SFE), and ultrasound‐assisted extraction (UAE).     

Multivariate optimisation of microwave-assisted extraction of capsaicin from Capsicum frutescens L. and quantitative analysis by 1H-NMR

A simple and rapid microwave-assisted extraction (MAE) procedure combined with 1H-NMR spectrometry was developed and optimised for the extraction and quantitative determination of capsaicin in Capsicum frutescens. The influence of experimental variables, including irradiation power, extraction temperature and dynamic extraction time before reaching the selected extraction temperature, on the performance of the extraction procedure was systematically studied using a Box-Behnken experimental design followed by a conventional central composite design approach. Statistical treatment of the results together with results from some additional experiments suggested optimum extraction conditions as 120 degrees C and 150 W, for 15 min with acetone as extractant. The optimised MAE method provides extracts that can be analysed quantitatively using 1H-NMR without any preliminary clean-up or derivatisation steps. In the 1H-NMR spectrum of the crude extracts the doublet signal in the delta range 4.349-4.360 ppm was well separated from other resonances in deuterated chloroform. The quantity of the compound was calculated from the relative ratio of the integral value of the target peak to that of a known amount of dimethylformamide as internal standard. In comparison with traditional Soxhlet extraction, the proposed method is less labour-intensive and provides a drastic reduction of extraction time and solvent consumption. In addition, MAE showed higher extraction yield and selectivity, with comparable reproducibility and recovery, relative to both conventional Soxhlet and sonication methods.     

微波辅助提取荔枝核黄酮类化合物及其抗氧化性研究

研究微波辅助法提取荔枝核黄酮类化合物的工艺,考察了提取溶剂、微波功率、溶剂用量、辐射时间、提取次数等因素对提取的影响。通过正交实验确定最佳的提取参数为:60%乙醇作为提取溶剂,微波功率700 W,料液比1∶25,辐射时间150 s,提取一次。在此优化条件下用微波辅助,黄酮类化合物的得率为6.86%,提取物中黄酮含量达到36.7%。抗氧化性研究表明荔枝核黄酮类化合物有良好的抗氧化活性,能有效清除羟基自由基(OH.)和超氧阴离子自由基(O2-.)。     

Determination of PAEs in soil of municipal wards of Patna,India by microwave assisted extraction and LC-MS/MS

Phthalic Acid Esters (PAEs) have become ubiquitous in environment due to unrestrained manufacture and use of plasticizers. The occurrence of PAEs in food, beverages, environmental and biological samples has been reported worldwide. PAEs are widely used in plasticizers for imparting flexibility and are not covalently bonded with the polymers, enabling PAEs to leach out due to change in pH and temperature. This study attempts to identify and quantify the PAEs in soil of Patna, India. Soil samples were collected from 22 municipal wards of Patna, Bihar. Microwave-Assisted Extraction (MAE) technique was applied for the extraction of PAEs followed by LC-MS/MS with electrospray ionization for the identification of different PAEs. Mono(4-pentenyl)phthalate (M(4P)P), Bis(2-hydroxyethyl) phthalate (BHEP), 2-Methylbutyl benzyl Phthalate (MBBP), Octyl Decyl phthalate (ODP), Heptadecyl trimethylsilyl phthalate (HDTMP), Magnesium phthalate (MgP), Diethyl Phthalate (DEP) and Dibutyl phthalate (DBP) were identified by LC-MS/MS. Interestingly, it was observed that soil samples of nine municipal wards were contaminated with high levels of maximum number PAEs identified in the study. Conclusively, the assessment of PAEs in soils samples is imperative for the management of environmental pollution generated by human activity.     

Evaluation of the Soil Contamination of Tangier (Morocco) by the Determination of BTEX,PCBs, and PAHs

Benzene, toluene, ethylbenzene, and xylenes (BTEX), twelve polycyclic aromatic hydrocarbons (PAHs) and seven polychlorinated biphenyls (PCBs), were selected as pollutants to evaluate the contamination of soils in the urban and industrial areas of Tangier (Morocco). PAHs and PCBs were determined by gas chromatography-mass spectrometry (GC-MS) after a microwave-assisted extraction (MAE) and gel permeation chromatography (GPC) clean-up. BTEX were directly determined by head-space GC-MS. Results obtained in this study show the presence of high levels of BTEX and PAHs in the soil near the urban waste deposit. However, the analysis of pollutants in the other sampling sites provided comprehensive evidence that soils of Tangier city are not contaminated.