Stepwise dissection and visualization of the catalytic mechanism of haloalkane dehalogenase LinB using molecular dynamics simulations and computer graphics

The different steps of the dehalogenation reaction carried out by LinB on three different substrates have been characterized using a combination of quantum mechanical calculations and molecular dynamics simulations. This has allowed us to obtain information in atomic detail about each step of the reaction mechanism, that is, substrate entrance and achievement of the near-attack conformation, transition state stabilization within the active site, halide stabilization, water molecule activation and subsequent hydrolytic attack on the ester intermediate with formation of alcohol, and finally product release. Importantly, no bias or external forces were applied during the whole procedure so that both intermediates and products were completely free to sample configuration space in order to adapt to the plasticity of the active site and/or search for an exit. Differences in substrate reactivity were found to be correlated with the ease of adopting the near-attack conformation and two different exit pathways were found for product release that do not interfere with substrate entrance. Additional support for the different entry and exit pathways was independently obtained from an examination of the enzyme's normal modes.     

Analysis of the reaction mechanism and substrate specificity of haloalkane dehalogenases by sequential and structural comparisons

Haloalkane dehalogenases catalyse environmentally importantdehalogenation reactions. These microbial enzymes representobjects of interest for protein engineering studies, attemptingto improve their catalytic efficiency or broaden their substratespecificity towards environmental pollutants. This paper presentsthe results of a comparative study of haloalkane dehalogenasesoriginating from different organisms. Protein sequences andthe models of tertiary structures of haloalkane dehalogenaseswere compared to investigate the protein fold, reaction mechanismand substrate specificity of these enzymes. Haloalkane dehalogenasescontain the structural motifs of /ß-hydrolases and epoxidaseswithin their sequences. They contain a catalytic triad withtwo different topological arrangements. The presence of a structurallyconserved oxyanion hole suggests the two-step reaction mechanismpreviously described for haloalkane dehalogenase from Xanthobacterautotrophicus GJ10. The differences in substrate specificityof haloalkane dehalogenases originating from different speciesmight be related to the size and geometry of an active siteand its entrance and the efficiency of the transition stateand halide ion stabilization by active site residues. Structurallyconserved motifs identified within the sequences can be usedfor the design of specific primers for the experimental screeningof haloalkane dehalogenases. Those amino acids which were predictedto be functionally important represent possible targets forfuture site-directed mutagenesis experiments.     

Comparison of the QSAR models for toxicity and biodegradability of anilines and phenols

Structure-activity models for toxicity and biodegradability of groups of m-anilines and p-phenols were developed and compared. Hydrophobicity was the most important property in determining toxicity. Whereas, electronic and steric properties were the more important in modeling biodegradation.     

Expansion of Access Tunnels and Active‐Site Cavities Influence Activity of Haloalkane Dehalogenases in Organic Cosolvents

The use of enzymes for biocatalysis can be significantly enhanced by using organic cosolvents in the reaction mixtures. Selection of the cosolvent type and concentration range for an enzymatic reaction is challenging and requires extensive empirical testing. An understanding of protein–solvent interaction could provide a theoretical framework for rationalising the selection process. Here, the behaviour of three model enzymes (haloalkane dehalogenases) was investigated in the presence of three representative organic cosolvents (acetone, formamide, and isopropanol). Steady‐state kinetics assays, molecular dynamics simulations, and time‐resolved fluorescence spectroscopy were used to elucidate the molecular mechanisms of enzyme–solvent interactions. Cosolvent molecules entered the enzymes' access tunnels and active sites, enlarged their volumes with no change in overall protein structure, but surprisingly did not act as competitive inhibitors. At low concentrations, the cosolvents either enhanced catalysis by lowering K_0.5 and increasing k_cat, or caused enzyme inactivation by promoting substrate inhibition and decreasing k_cat. The induced activation and inhibition of the enzymes correlated with expansion of the active‐site pockets and their occupancy by cosolvent molecules. The study demonstrates that quantitative analysis of the proportions of the access tunnels and active‐sites occupied by organic solvent molecules provides the valuable information for rational selection of appropriate protein–solvent pair and effective cosolvent concentration.     

Structural Analysis of the Ancestral Haloalkane Dehalogenase AncLinB-DmbA

Haloalkane dehalogenases (EC 3.8.1.5) play an important role in hydrolytic degradation of halogenated compounds, resulting in a halide ion, a proton, and an alcohol. They are used in biocatalysis, bioremediation, and biosensing of environmental pollutants and also for molecular tagging in cell biology. The method of ancestral sequence reconstruction leads to prediction of sequences of ancestral enzymes allowing their experimental characterization. Based on the sequences of modern haloalkane dehalogenases from the subfamily II, the most common ancestor of thoroughly characterized enzymes LinB from Sphingobium japonicum UT26 and DmbA from Mycobacterium bovis 5033/66 was in silico predicted, recombinantly produced and structurally characterized. The ancestral enzyme AncLinB-DmbA was crystallized using the sitting-drop vapor-diffusion method, yielding rod-like crystals that diffracted X-rays to 1.5 Å resolution. Structural comparison of AncLinB-DmbA with their closely related descendants LinB and DmbA revealed some differences in overall structure and tunnel architecture. Newly prepared AncLinB-DmbA has the highest active site cavity volume and the biggest entrance radius on the main tunnel in comparison to descendant enzymes. Ancestral sequence reconstruction is a powerful technique to study molecular evolution and design robust proteins for enzyme technologies.     

Computational site-directed mutagenesis of haloalkane dehalogenase in position 172

Laparoscopic cholecystectomy is considered as the new gold standard operation for removal of the gallbladder, and has several advantages over the traditional open cholecystectomy. However, in the last few years there is an increasing number of case reports of port site metastases following laparoscopic cholecystectomy for unsuspected carcinoma of the gallbladder. Two case reports of trocar site metastases are presented, and they further highlight the concern of the role of minimal invasive surgery in the presence of unsuspected carcinoma of the gallbladder. In this review we speculate on the mechanisms which may be responsible for metastatic deposits during laparoscopic cholecystectomy and suggest certain recommendations.     

Ancestral Haloalkane Dehalogenases Show Robustness and Unique Substrate Specificity

Ancestral sequence reconstruction (ASR) represents a powerful approach for empirical testing structure‐function relationships of diverse proteins. We employed ASR to predict sequences of five ancestral haloalkane dehalogenases (HLDs) from the HLD‐II subfamily. Genes encoding the inferred ancestral sequences were synthesized and expressed in Escherichia coli, and the resurrected ancestral enzymes (AncHLD1–5) were experimentally characterized. Strikingly, the ancestral HLDs exhibited significantly enhanced thermodynamic stability compared to extant enzymes (ΔT_m up to 24 °C), as well as higher specific activities with preference for short multi‐substituted halogenated substrates. Moreover, multivariate statistical analysis revealed a shift in the substrate specificity profiles of AncHLD1 and AncHLD2. This is extremely difficult to achieve by rational protein engineering. The study highlights that ASR is an efficient approach for the development of novel biocatalysts and robust templates for directed evolution.     

Domestic infestation by Triatoma infestans and prevalence of Trypanosoma cruzi seropositives in a rural area of the Argentinian northeast

An epidemiological study was carried out from April 1991 to December 1993 to obtain a general view of T. cruzi domestic transmission in rural areas of San Miguei Department, Corrientes. From 100 analyzed households, 50.0% was infested by T infestans (Klug, 1834) and 1.0% by T. sordida (Stal, 1859). Domiciliary colonization by T. sordida is reported for first time in Corrientes province. T. cruzi infection of T. infestans was 23.1%. Indirect hemagglutination and indirect immunofluorescence antibody tests were used for detection of anti T. cruzi antibodies in 388 human sera, 23.4% showed serological reactivity. An important high rate (12.9%) was observed in the age group of higher transmission risk. Seropositives percentages increased with age and reached 50.0% in 31-40 years old group. Domestic infestation by T. infestans, seropositive human prevalence to T. cruzi and householders precarious life conditions prove that this endemic disease is still a problem in the studied area.