Zbyněk Prokop

Mobility, bioavailability, and toxic effects of cadmium in soil samples

Total concentration is not a reliable indicator of metal mobility or bioavailability in soils. The physicochemical form determines the behavior of metals in soils and hence the toxicity toward terrestrial biota. The main objectives of this study were the application and comparison of three approaches for the evaluation of cadmium behavior in soil samples. The mobility and bioavailability of cadmium in five selected soil samples were evaluated using equilibrium speciation (Windermere humic aqueous model (WHAM)), extraction procedures (Milli-Q water, DMSO, and DTPA), and a number of bioassays (Microtox, growth inhibition test, contact toxicity test, and respiration). The mobility, represented by the water-extractable fraction, corresponded well with the amount of cadmium in the soil solution, calculated using the WHAM (r(2)=0.96, P<0.001). The results of the ecotoxicological evaluation, which represent the bioavailable fraction of cadmium, correlated well with DTPA extractability and also with the concentration of free cadmium ion, which is recognized as the most bioavailable metal form. The results of the WHAM as well as the results of extraction experiments showed a strong binding of cadmium to organic matter and a weak sorption of cadmium to clay minerals.

A Single Mutation in a Tunnel to the Active Site Changes the Mechanism and Kinetics of Product Release in Haloalkane Dehalogenase LinB

Background: Tunnel properties affect ligand passage in enzymes with buried active sites. Results: A tunnel mutation from leucine to tryptophan changes the mechanism of bromide ion release from haloalkane dehalogenase LinB. Conclusion: Interactions of the bromide ion with the tryptophan increase free energy barrier for its passage, causing the reaction mechanism change. Significance: The results provide guidelines for enzyme engineering. Many enzymes have buried active sites. The properties of the tunnels connecting the active site with bulk solvent affect ligand binding and unbinding and also the catalytic properties. Here, we investigate ligand passage in the haloalkane dehalogenase enzyme LinB and the effect of replacing leucine by a bulky tryptophan at a tunnel-lining position. Transient kinetic experiments show that the mutation significantly slows down the rate of product release. Moreover, the mechanism of bromide ion release is changed from a one-step process in the wild type enzyme to a two-step process in the mutant. The rate constant of bromide ion release corresponds to the overall steady-state turnover rate constant, suggesting that product release became the rate-limiting step of catalysis in the mutant. We explain the experimental findings by investigating the molecular details of the process computationally. Analysis of trajectories from molecular dynamics simulations with a tunnel detection software reveals differences in the tunnels available for ligand egress. Corresponding differences are seen in simulations of product egress using a specialized enhanced sampling technique. The differences in the free energy barriers for egress of a bromide ion obtained using potential of mean force calculations are in good agreement with the differences in rates obtained from the transient kinetic experiments. Interactions of the bromide ion with the introduced tryptophan are shown to affect the free energy barrier for its passage. The study demonstrates how the mechanism of an enzymatic catalytic cycle and reaction kinetics can be engineered by modification of protein tunnels.

Weak Activity of Haloalkane Dehalogenase Linb with 1,2,3-Trichloropropane Revealed by X-Ray Crystallography and Microcalorimetry

ABSTRACT 1,2,3-Trichloropropane (TCP) is a highly toxic and recalcitrant compound. Haloalkane dehalogenases are bacterial enzymes that catalyze the cleavage of a carbon-halogen bond in a wide range of organic halogenated compounds. Haloalkane dehalogenase LinB from Sphingobium japonicum UT26 has, for a long time, been considered inactive with TCP, since the reaction cannot be easily detected by conventional analytical methods. Here we demonstrate detection of the weak activity (kcat = 0.005 s−1) of LinB with TCP using X-ray crystallography and microcalorimetry. This observation makes LinB a useful starting material for the development of a new biocatalyst toward TCP by protein engineering. Microcalorimetry is proposed to be a universal method for the detection of weak enzymatic activities. Detection of these activities is becoming increasingly important for engineering novel biocatalysts using the scaffolds of proteins with promiscuous activities.

Reconstruction of Mycobacterial Dehalogenase Rv2579 by Cumulative Mutagenesis of Haloalkane Dehalogenase LinB

ABSTRACT The homology model of protein Rv2579 from Mycobacterium tuberculosis H37Rv was compared with the crystal structure of haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26, and this analysis revealed that 6 of 19 amino acid residues which form an active site and entrance tunnel are different in LinB and Rv2579. To characterize the effect of replacement of these six amino acid residues, mutations were introduced cumulatively into the six amino acid residues of LinB. The sixfold mutant, which was supposed to have the active site of Rv2579, exhibited haloalkane dehalogenase activity with the haloalkanes tested, confirming that Rv2579 is a member of the haloalkane dehalogenase protein family.

Immobilization of Haloalkane Dehalogenase LinB from Sphingobium japonicum UT26 for Biotechnological Applications

Immobilization of Haloalkane dehalogenase LinB from Sphingobium japonicum UT26 for Biotechnological Applications Haloalkane dehalogenases are enzymes capable of converting a broad range of aliphatic halogenated compounds to corresponding alcohols. These dehalogenase-based biotransformations are attractive for various biological processes, e.g. biocatalysis, bioremediation and detoxification, which often require protein immobilization.

Bioavailability and mobility of organic contaminants in soil: new three-step ecotoxicological evaluation.

AbstractA novel approach was developed for rapid assessment of bioavailability and potential mobility of contaminants in soil. The response of the same test organism to the organic extract, water extract and solid phase of soil was recorded and compared. This approach was designed to give an initial estimate of the total organic toxicity (response to organic extractable fraction), as well as the mobile (response to water extract) and bioavailable fraction (response to solid phase) of soil samples. Eighteen soil samples with different levels of pollution and content of organic carbon were selected to validate the novel three-step ecotoxicological evaluation approach. All samples were chemically analysed for priority contaminants, including aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), hexachlorocyclohexane (HCH) and dichlordiphenyltrichloroethane (DDT). The ecotoxicological evaluation involved determination of toxicity of the organic, mobile and bioavailable fractions of soil to the test organism, bacterium Bacillus cereus. We found a good correlation between the chemical analysis and the toxicity of organic extract. The low toxicity of water extracts indicated low water solubility, and thus, low potential mobility of toxic contaminants present in the soil samples. The toxicity of the bioavailable fraction was significantly greater than the toxicity of water-soluble (mobile) fraction of the contaminants as deduced from comparing untreated samples and water extracts. The bioavailability of the contaminants decreased with increasing concentrations of organic carbon in evaluated soil samples. In conclusion, the three-step ecotoxicological evaluation utilised in this study can give a quick insight into soil contamination in context with bioavailability and mobility of the contaminants present. This information can be useful for hazard identification and risk assessment of soil-associated contaminants. Graphical AbstractNew three-step ecotoxicological evaluation by using the same organism

The use of a microbial contact toxicity test for evaluating cadmium bioavailability in soil

Background, Aims and ScopeBioavailability of toxic compounds in soil can be defined as the fraction able to come into contact with biota and to cause toxic effects. The contact toxicity tests may detect the total toxic response of all bioavailable contaminants present in a sample. The objectives of this study were to evaluate the use of microbial contact toxicity tests for cadmium bioavailability assessment and to evaluate the relationship between sorption, soil characteristics and cadmium bioavailability.MethodsA test soil bacterium,Bacillus cereus, was put in direct contact with the solid sample. Four unpolluted soils were selected to provide solid samples with a variety of physicochemical characteristics. The toxicity and sorption behaviour of cadmium spiked to the soil samples were determined.Results, Discussion and ConclusionsA significant correlation between contact toxicity test results and partitioning of cadmium in the soil samples (r2= 0.79, p <0.05; n = 26) was found. The results confirm that the bioavailability of cadmium in soil depends on its sorption behaviour. Cadmium sorbed to the cation exchange sites associated with fulvic acids is non-bioavailable in the toxicity test employed in this study. It is concluded that the microbial contact toxicity test is a suitable tool for detecting cadmium bioavailablity in the soils used in this study.OutlookThe application of microbial contact toxicity tests for bioavailability assessment can be very useful for the risk identification and remediation of soil-associated contaminants.

FireProt: web server for automated design of thermostable proteins

Abstract There is a continuous interest in increasing proteins stability to enhance their usability in numerous biomedical and biotechnological applications. A number of in silico tools for the prediction of the effect of mutations on protein stability have been developed recently. However, only single-point mutations with a small effect on protein stability are typically predicted with the existing tools and have to be followed by laborious protein expression, purification, and characterization. Here, we present FireProt, a web server for the automated design of multiple-point thermostable mutant proteins that combines structural and evolutionary information in its calculation core. FireProt utilizes sixteen tools and three protein engineering strategies for making reliable protein designs. The server is complemented with interactive, easy-to-use interface that allows users to directly analyze and optionally modify designed thermostable mutants. FireProt is freely available at http://loschmidt.chemi.muni.cz/fireprot.

The dynamics of xylem sap pH under drought: a universal response in herbs?

Background and aimsLong distance signals in xylem from roots to leaves are important in plant response to drought stress. Abscisic acid (ABA) plays a key role in drought signaling in plants but apoplastic pH may modulate its effect by distributing ABA into various compartments in leaves. We aimed to reveal the dynamics of changes in sap pH and its relationships with the transport of inorganic and organic ions in eight herbaceous plant species under continuously declining soil water content. We tested several hypotheses related to the mechanism of pH changes in xylem.MethodsWe used a pressure chamber to collect xylem sap and to measure of leaf/stem water potential at various stages of soil drying. We measured pH and concentrations of the most abundant inorganic (NO3−, SO42−, PO43− and Cl−) and organic (malate and citrate) anions in xylem sap.ResultsSpecies differed considerably in the dynamics of pH changes in xylem in drying soil. Changes in xylem sap pH during drying did not relate to the nitrogen assimilation strategy but may be affected by sap flow rate. Simultaneous changes in the concentrations of inorganic and organic anions were highly species-specific.ConclusionsHigh variability among species in the observed relationships in response to drought indicates that comparisons among different studies and the generalization of results should be made with caution.

Computer-assisted engineering of hyperstable fibroblast growth factor 2

Fibroblast growth factors (FGFs) serve numerous regulatory functions in complex organisms, and their corresponding therapeutic potential is of growing interest to academics and industrial researchers alike. However, applications of these proteins are limited due to their low stability. Here we tackle this problem using a generalizable computer‐assisted protein engineering strategy to create a unique modified FGF2 with nine mutations displaying unprecedented stability and uncompromised biological function. The data from the characterization of stabilized FGF2 showed a remarkable prediction potential of in silico methods and provided insight into the unfolding mechanism of the protein. The molecule holds a considerable promise for stem cell research and medical or pharmaceutical applications.