Metagenomic analysis of hot spring soil for mining a novel thermostable enzybiotic.

The misuse and overuse of antibiotics have contributed to a rapid emergence of antibiotic-resistant bacterial pathogens. This global health threat underlines the urgent need for innovative and novel antimicrobials. Endolysins derived from bacteriophages or prophages constitute promising new antimicrobials (so-called enzybiotics), exhibiting the ability to break down bacterial peptidoglycan (PG). In the present work, metagenomic analysis of soil samples, collected from thermal springs, allowed the identification of a prophage-derived endolysin that belongs to the N-acetylmuramoyl-L-alanine amidase type 2 (NALAA-2) family and possesses a LysM (lysin motif) region as a cell wall binding domain (CWBD). The enzyme (Ami1) was cloned and expressed in Escherichia coli, and its bactericidal and lytic activity was characterized. The results indicate that Ami1 exhibits strong bactericidal and antimicrobial activity against a broad range of bacterial pathogens, as well as against isolated peptidoglycan (PG). Among the examined bacterial pathogens, Ami1 showed highest bactericidal activity against Staphylococcus aureus sand Staphylococcus epidermidis cells. Thermostability analysis revealed a melting temperature of 64.2 ± 0.6 °C. Overall, these findings support the potential that Ami1, as a broad spectrum antimicrobial agent, could be further assessed as enzybiotic for the effective treatment of bacterial infections. KEY POINTS: • Metagenomic analysis allowed the identification of a novel prophage endolysin • The endolysin belongs to type 2 amidase family with lysin motif region • The endolysin displays high thermostability and broad bactericidal spectrum.

Characterization and Engineering Studies of a New Endolysin from the Propionibacterium acnes Bacteriophage PAC1 for the Development of a Broad-Spectrum Artilysin with Altered Specificity.

The emergence of multidrug-resistant (MDR) bacteria has risen rapidly, leading to a great threat to global public health. A promising solution to this problem is the exploitation of phage endolysins. In the present study, a putative N-acetylmuramoyl-L-alanine type-2 amidase (NALAA-2, EC 3.5.1.28) from Propionibacterium bacteriophage PAC1 was characterized. The enzyme (PaAmi1) was cloned into a T7 expression vector and expressed in E. coli BL21 cells. Kinetics analysis using turbidity reduction assays allowed the determination of the optimal conditions for lytic activity against a range of Gram-positive and negative human pathogens. The peptidoglycan degradation activity of PaAmi1 was confirmed using isolated peptidoglycan from P. acnes. The antibacterial activity of PaAmi1 was investigated using live P. acnes cells growing on agar plates. Two engineered variants of PaAmi1 were designed by fusion to its N-terminus two short antimicrobial peptides (AMPs). One AMP was selected by searching the genomes of Propionibacterium bacteriophages using bioinformatics tools, whereas the other AMP sequence was selected from the antimicrobial peptide databases. Both engineered variants exhibited improved lytic activity towards P. acnes and the enterococci species Enterococcus faecalis and Enterococcus faecium. The results of the present study suggest that PaAmi1 is a new antimicrobial agent and provide proof of concept that bacteriophage genomes are a rich source of AMP sequences that can be further exploited for designing novel or improved endolysins.

A Key Role in Catalysis and Enzyme Thermostability of a Conserved Helix H5 Motif of Human Glutathione Transferase A1-1.

Glutathione transferases (GSTs) are promiscuous enzymes whose main function is the detoxification of electrophilic compounds. These enzymes are characterized by structural modularity that underpins their exploitation as dynamic scaffolds for engineering enzyme variants, with customized catalytic and structural properties. In the present work, multiple sequence alignment of the alpha class GSTs allowed the identification of three conserved residues (E137, K141, and S142) at α-helix 5 (H5). A motif-directed redesign of the human glutathione transferase A1-1 (hGSTA1-1) was performed through site-directed mutagenesis at these sites, creating two single- and two double-point mutants (E137H, K141H, K141H/S142H, and E137H/K141H). The results showed that all the enzyme variants displayed enhanced catalytic activity compared to the wild-type enzyme hGSTA1-1, while the double mutant hGSTA1-K141H/S142H also showed improved thermal stability. X-ray crystallographic analysis revealed the molecular basis of the effects of double mutations on enzyme stability and catalysis. The biochemical and structural analysis presented here will contribute to a deeper understanding of the structure and function of alpha class GSTs.

Structural Studies of Klebsiella pneumoniae Fosfomycin-Resistance Protein and Its Application for the Development of an Optical Biosensor for Fosfomycin Determination.

Fosfomycin-resistance proteins (FosAs) are dimeric metal-dependent glutathione transferases that conjugate the antibiotic fosfomycin (Fos) to the tripeptide glutathione (γ-Glu-Cys-Gly, GSH), rendering it inactive. In the present study, we reported a comparative analysis of the functional features of two FosAs from Pseudomonas aeruginosa (FosAPA) and Klebsiella pneumoniae (FosAKP). The coding sequences of the enzymes were cloned into a T7 expression vector, and soluble active enzymes were expressed in E. coli. FosAKP displayed higher activity and was selected for further studies. The crystal structure of the dimeric FosAKP was determined via X-ray crystallography at 1.48 Šresolution. Fos and tartrate (Tar) were found bound in the active site of the first and second molecules of the dimer, respectively. The binding of Tar to the active site caused slight rearrangements in the structure and dynamics of the enzyme, acting as a weak inhibitor of Fos binding. Differential scanning fluorimetry (DSF) was used to measure the thermal stability of FosAKP under different conditions, allowing for the selection of a suitable buffer to maximize enzyme operational stability. FosAKP displays absolute specificity towards Fos; therefore, this enzyme was exploited for the development of an enzyme-based colorimetric biosensor. FosAKP was tethered at the bottom of a plastic cuvette using glutaraldehyde chemistry to develop a simple colorimetric method for the determination of Fos in drinking water and animal plasma.

Directed Evolution of Phi Class Glutathione Transferases Involved in Multiple-Herbicide Resistance of Grass Weeds and Crops.

The extensive application of herbicides in crop cultivation has indisputably led to the emergence of weed populations characterized by multiple herbicide resistance (MHR). This phenomenon is associated with the enhanced metabolism and detoxifying ability of endogenous enzymes, such as phi class glutathione transferases (GSTFs). In the present work, a library of mutant GSTFs was created by in vitro directed evolution via DNA shuffling. Selected gstf genes from the weeds Alopecurus myosuroides and Lolium rigidum, and the cereal crops Triticum durum and Hordeum vulgare were recombined to forge a library of novel chimeric GSTFs. The library was activity screened and the best-performing enzyme variants were purified and characterized. The work allowed the identification of enzyme variants that exhibit an eight-fold improvement in their catalytic efficiency, higher thermal stability (8.3 °C) and three-times higher inhibition sensitivity towards the herbicide butachlor. The crystal structures of the best-performing enzyme variants were determined by X-ray crystallography. Structural analysis allowed the identification of specific structural elements that are responsible for kcat regulation, thermal stability and inhibition potency. These improved novel enzymes hold the potential for utilization in biocatalysis and green biotechnology applications. The results of the present work contribute significantly to our knowledge of the structure and function of phi class plant GSTs and shed light on their involvement in the mechanisms of MHR.

Ligandability Assessment of Human Glutathione Transferase M1-1 Using Pesticides as Chemical Probes.

Glutathione transferases (GSTs; EC 2.5.1.18) form a group of multifunctional enzymes that are involved in phase II of the cellular detoxification mechanism and are associated with increased susceptibility to cancer development and resistance to anticancer drugs. The present study aims to evaluate the ligandability of the human GSTM1-1 isoenzyme (hGSTM1-1) using a broad range of structurally diverse pesticides as probes. The results revealed that hGSTM1-1, compared to other classes of GSTs, displays limited ligandability and ligand-binding promiscuity, as revealed by kinetic inhibition studies. Among all tested pesticides, the carbamate insecticide pirimicarb was identified as the strongest inhibitor towards hGSTM1-1. Kinetic inhibition analysis showed that pirimicarb behaved as a mixed-type inhibitor toward glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB). To shine a light on the restricted hGSTM1-1 ligand-binding promiscuity, the ligand-free crystal structure of hGSTM1-1 was determined by X-ray crystallography at 1.59 Å-resolution. Comparative analysis of ligand-free structure with the available ligand-bound structures allowed for the study of the enzyme's plasticity and the induced-fit mechanism operated by hGSTM1-1. The results revealed important structural features of the H-site that contribute to xenobiotic-ligand binding and specificity. It was concluded that hGSTM1-1 interacts preferentially with one-ring aromatic compounds that bind at a discrete site which partially overlaps with the xenobiotic substrate binding site (H-site). The results of the study form a basis for the rational design of new drugs targeting hGSTM1-1.

Robotics for enzyme technology: innovations and technological perspectives.

The use of robotics in the life science sector has created a considerable and significant impact on a wide range of research areas, including enzyme technology due to their immense applications in enzyme and microbial engineering as an indispensable tool in high-throughput screening applications. Scientists are experiencing the advanced applications of various biological robots (nanobots), fabricated based on bottom-up or top-down approaches for making nanotechnology scaffolds. Nanobots and enzyme-powered nanomotors are particularly attractive because they are self-propelled vehicles, which consume biocompatible fuels. These smart nanostructures are widely used as drug delivery systems for the efficient treatment of various diseases. This review gives insights into the escalating necessity of robotics and nanobots and their ever-widening applications in enzyme technology, including biofuel production and biomedical applications. It also offers brief insights into high-throughput robotic platforms that are currently being used in enzyme screening applications for monitoring and control of microbial growth conditions. KEY POINTS: • Robotics and their applications in biotechnology are highlighted. • Robotics for high-throughput enzyme screening and microbial engineering are described. • Nanobots and enzyme-powered nanomotors as controllable drug delivery systems are reviewed.

Overexpression of A Biotic Stress-Inducible Pvgstu Gene Activates Early Protective Responses in Tobacco under Combined Heat and Drought.

Drought and heat stresses are major factors limiting crop growth and productivity, and their effect is more devastating when occurring concurrently. Plant glutathione transferases (GSTs) are differentially expressed in response to different stimuli, conferring tolerance to a wide range of abiotic stresses. GSTs from drought-tolerant Phaseolus vulgaris var. "Plake Megalosperma Prespon" is expected to play an important role in the response mechanisms to combined and single heat and drought stresses. Herein, we examined wild-type N. tabacum plants (cv. Basmas Xanthi) and T1 transgenic lines overexpressing the stress-induced Pvgstu3-3 and Pvgstu2-2 genes. The overexpression of Pvgstu3-3 contributed to potential thermotolerance and greater plant performance under combined stress. Significant alterations in the primary metabolism were observed in the transgenic plants between combined stress and stress-free conditions. Stress-responsive differentially expressed genes (DEGs) and transcription factors (TFs) related to photosynthesis, signal transduction, starch and sucrose metabolism, osmotic adjustment and thermotolerance, were identified under combined stress. In contrast, induction of certain DEGs and TF families under stress-free conditions indicated that transgenic plants were in a primed state. The overexpression of the Pvgstu3-3 is playing a leading role in the production of signaling molecules, induction of specific metabolites and activation of the protective mechanisms for enhanced protection against combined abiotic stresses in tobacco.

The Interaction of Human Glutathione Transferase GSTA1-1 with Reactive Dyes.

Human glutathione transferase A1-1 (hGSTA1-1) contributes to developing resistance to anticancer drugs and, therefore, is promising in terms of drug-design targets for coping with this phenomenon. In the present study, the interaction of anthraquinone and diazo dichlorotriazine dyes (DCTD) with hGSTA1-1 was investigated. The anthraquinone dye Procion blue MX-R (PBMX-R) appeared to interact with higher affinity and was selected for further study. The enzyme was specifically and irreversibly inactivated by PBMX-R, following a biphasic pseudo-first-order saturation kinetics, with approximately 1 mol of inhibitor per mol of the dimeric enzyme being incorporated. Molecular modeling and protein chemistry data suggested that the modified residue is the Cys112, which is located at the entrance of the solvent channel at the subunits interface. The results suggest that negative cooperativity exists upon PBMX-R binding, indicating a structural communication between the two subunits. Kinetic inhibition analysis showed that the dye is a competitive inhibitor towards glutathione (GSH) and mixed-type inhibitor towards 1-chloro-2,4-dinitrobenzene (CDNB). The present study results suggest that PBMX-R is a useful probe suitable for assessing by kinetic means the drugability of the enzyme in future drug-design efforts.

Probing the Role of the Conserved Arg174 in Formate Dehydrogenase by Chemical Modification and Site-Directed Mutagenesis.

The reactive adenosine derivative, adenosine 5'-O-[S-(4-hydroxy-2,3-dioxobutyl)]-thiophosphate (AMPS-HDB), contains a dicarbonyl group linked to the purine nucleotide at a position equivalent to the pyrophosphate region of NAD+. AMPS-HDB was used as a chemical label towards Candida boidinii formate dehydrogenase (CbFDH). AMPS-HDB reacts covalently with CbFDH, leading to complete inactivation of the enzyme activity. The inactivation kinetics of CbFDH fit the Kitz and Wilson model for time-dependent, irreversible inhibition (KD = 0.66 ± 0.15 mM, first order maximum rate constant k3 = 0.198 ± 0.06 min-1). NAD+ and NADH protects CbFDH from inactivation by AMPS-HDB, showing the specificity of the reaction. Molecular modelling studies revealed Arg174 as a candidate residue able to be modified by the dicarbonyl group of AMPS-HDB. Arg174 is a strictly conserved residue among FDHs and is located at the Rossmann fold, the common mononucleotide-binding motif of dehydrogenases. Arg174 was replaced by Asn, using site-directed mutagenesis. The mutant enzyme CbFDHArg174Asn was showed to be resistant to inactivation by AMPS-HDB, confirming that the guanidinium group of Arg174 is the target for AMPS-HDB. The CbFDHArg174Asn mutant enzyme exhibited substantial reduced affinity for NAD+ and lower thermostability. The results of the study underline the pivotal and multifunctional role of Arg174 in catalysis, coenzyme binding and structural stability of CbFDH.

Delineation of the functional and structural properties of the glutathione transferase family from the plant pathogen Erwinia carotovora.

Erwinia carotovora, a widespread plant pathogen that causes soft rot disease in many plants, is considered a major threat in agriculture. Bacterial glutathione transferases (GSTs) play important roles in a variety of metabolic pathways and processes, such as the biodegradation of xenobiotics, protection against abiotic stress, and resistance against antimicrobial drugs. The GST family of canonical soluble enzymes from Erwinia carotovora subsp. atroseptica strain SCRI1043 (EcaGSTs) was investigated. Genome analysis showed the presence of six putative canonical cytoplasmic EcaGSTs, which were revealed by phylogenetic analysis to belong to the well-characterized GST classes beta, nu, phi, and zeta. The analysis also revealed the presence of two isoenzymes that were phylogenetically close to the omega class of GSTs, but formed a distinct class. The EcaGSTs were cloned and expressed in Escherichia coli, and their catalytic activity toward different electrophilic substrates was elucidated. The EcaGSTs catalyzed different types of reactions, although all enzymes were particularly active in reactions involving electrophile substitution. Gene and protein expression profiling conducted under normal culture conditions as well as in the presence of the herbicide alachlor and the xenobiotic 1-chloro-2,4-dinitrobenzene (CDNB) showed that the isoenzyme EcaGST1, belonging to the omega-like class, was specifically induced at both the protein and mRNA levels. EcaGST1 presumably participates in counteracting the xenobiotic toxicity and/or abiotic stress conditions, and may therefore represent a novel molecular target in the development of new chemical treatments to control soft rot diseases.

Bioprospecting of functional cellulases from metagenome for second generation biofuel production: a review.

Second generation biofuel production has been appeared as a sustainable and alternative energy option. The ultimate aim is the development of an industrially feasible and economic conversion process of lignocellulosic biomass into biofuel molecules. Since, cellulose is the most abundant biopolymer and also represented as the photosynthetically fixed form of carbon, the efficient hydrolysis of cellulose is the most important step towards the development of a sustainable biofuel production process. The enzymatic hydrolysis of cellulose by suites of hydrolytic enzymes underlines the importance of cellulase enzyme system in whole hydrolysis process. However, the selection of the suitable cellulolytic enzymes with enhanced activities remains a challenge for the biorefinery industry to obtain efficient enzymatic hydrolysis of biomass. The present review focuses on deciphering the novel and effective cellulases from different environmental niches by unculturable metagenomic approaches. Furthermore, a comprehensive functional aspect of cellulases is also presented and evaluated by assessing the structural and catalytic properties as well as sequence identities and expression patterns. This review summarizes the recent development in metagenomics based approaches for identifying and exploring novel cellulases which open new avenues for their successful application in biorefineries.

Recent advances in protein engineering and biotechnological applications of glutathione transferases.

Glutathione transferases (GSTs, EC 2.5.1.18) are a widespread family of enzymes that play a central role in the detoxification, metabolism, and transport or sequestration of endogenous or xenobiotic compounds. During the last two decades, delineation of the important structural and catalytic features of GSTs has laid the groundwork for engineering GSTs, involving both rational and random approaches, aiming to create new variants with new or altered properties. These approaches have expanded the usefulness of native GSTs, not only for understanding the fundamentals of molecular detoxification mechanisms, but also for the development medical, analytical, environmental, and agricultural applications. This review article attempts to summarize successful examples and current developments on GST engineering, highlighting in parallel the recent knowledge gained on their phylogenetic relationships, structural/catalytic features, and biotechnological applications.

Antioxidant capacity and immunomodulatory effects of a chrysolaminarin-enriched extract in Senegalese sole.

The microalgae are an important source of bioactive molecules including ß-glucans that can be used as immunostimulants in aquaculture. In the present study, the antioxidant capacity, cytotoxicity and immunomodulatory activity of a chrysolaminarin-enriched extract obtained from the diatom Phaeodactylum tricornutum was evaluated. The extract showed a higher total antioxidant activity as determined by ORAC and FRAP assays and a lower DPPH scavenging activity than particulate yeast-ß-glucan. The cytotoxicity test indicated that extract concentrations higher than 0.01% w/v could impair cell viability of human dermal fibroblasts. To evaluate the immunomodulatory activity, juvenile soles were intraperitoneally injected with the chrysolaminarin-enriched extract suspended in coconut oil (1 mg/fish) followed by a reinjection at 7 days. A sham group injected with the carrier solution was maintained as a negative control. Cumulated mortality of fish injected with the chrysolaminarin-enriched extract was 29.4% after six days and no mortality was recorded after extract reinjection. Expression analyses of fifteen genes related to the innate immune system in kidney, spleen and intestine showed temporal and organ-specific responses. A rapid (2 days post-injection; dpi) and strong induction of the pro-inflammatory il1b and the antimicrobial peptide hamp1 in the three immunological organs, the hsp90aa in kidney and spleen, irf3 in intestine and c3 in spleen was observed indicating a potent inflammatory response. The recovery of steady-state levels for all activated genes at 5 dpi, and the down-regulation of c-lectin receptor as well as some interferon-related genes (ifn1, irf1, irf3, irf8, irf9 and mx) in kidney and cxc10 in spleen indicated that the soles were able to activate a homeostatic response against the ß-glucan insult. The reinjection of the chrysolaminarin-enriched extract did not activate a new inflammatory response but reduced the mRNA levels of hsp90aa and irf3 indicating that soles developed some resistance to ß-glucans. Overall, these results reveal this enriched extract as a novel and potent source of ß-glucans with antioxidant and immunomodulatory capacity suitable for immunostimulation in aquaculture.

Molecular diversity and phylogeny of indigenous Rhizobium leguminosarum strains associated with Trifolium repens plants in Romania.

The symbiotic nitrogen fixing legumes play an essential role in sustainable agriculture. White clover (Trifolium repens L.) is one of the most valuable perennial legumes in pastures and meadows of temperate regions. Despite its great agriculture and economic importance, there is no detailed available information on phylogenetic assignation and characterization of rhizobia associated with native white clover plants in South-Eastern Europe. In the present work, the diversity of indigenous white clover rhizobia originating in 11 different natural ecosystems in North-Eastern Romania were assessed by a polyphasic approach. Initial grouping showed that, 73 rhizobial isolates, representing seven distinct phenons were distributed into 12 genotypes, indicating a wide phenotypic and genotypic diversity among the isolates. To clarify their phylogeny, 44 representative strains were used in sequence analysis of 16S rRNA gene and IGS fragments, three housekeeping genes (atpD, glnII and recA) and two symbiosis-related genes (nodA and nifH). Multilocus sequence analysis (MLSA) phylogeny based on concatenated housekeeping genes delineated the clover isolates into five putative genospecies. Despite their diverse chromosomal backgrounds, test strains shared highly similar symbiotic genes closely related to Rhizobium leguminosarum biovar trifolii. Phylogenies inferred from housekeeping genes were incongruent with those of symbiotic genes, probably due to occurrence of lateral transfer events among native strains. This is the first polyphasic taxonomic study to report on the MLSA-based phylogenetic diversity of indigenous rhizobia nodulating white clover plants grown in various soil types in South-Eastern Europe. Our results provide valuable taxonomic data on native clover rhizobia and may increase the pool of genetic material to be used as biofertilizers.

Insights into the Influence of Specific Splicing Events on the Structural Organization of LRRK2.

Leucine-rich repeat kinase 2 (LRRK2) is a large protein of unclear function. Rare mutations in the LRRK2 gene cause familial Parkinson's disease (PD) and inflammatory bowel disease. Genome-wide association studies (GWAS) have revealed significant association of the abovementioned diseases at the LRRK2 locus. Cell and systems biology research has led to potential roles that LRRK2 may have in PD pathogenesis, especially the kinase domain (KIN). Previous human expression studies showed evidence of mRNA expression and splicing patterns that may contribute to our understanding of the function of LRRK2. In this work, we investigate and identified significant regional differences in LRRK2 expression at the mRNA level, including a number of splicing events in the Ras of complex protein (Roc) and C-terminal of Roc domain (COR) of LRRK2, in the substantia nigra (SN) and occipital cortex (OCTX). Our findings indicate that the predominant form of LRRK2 mRNA is full length, with shorter isoforms present at a lower copy number. Our molecular modelling study suggests that splicing events in the ROC/COR domains will have major consequences on the enzymatic function and dimer formation of LRRK2. The implications of these are highly relevant to the broader effort to understand the biology and physiological functions of LRRK2, and to better characterize the role(s) of LRRK2 in the underlying mechanism leading to PD.

Delineation of the structural and functional role of Arg111 in GSTU4-4 from Glycine max by chemical modification and site-directed mutagenesis.

The structural and functional role of Arg111 in GSTU4-4 from Glycine max (GmGSTU4-4) was studied by chemical modification followed by site-directed mutagenesis. The arginine-specific reagent 2,3-butanedione (BTD) inactivates the enzyme in borate buffer at pH8.0, with pseudo-first-order saturation kinetics. The rate of inactivation exhibited a non-linear dependence on the concentration of BTD which can be described by reversible binding of reagent to the enzyme (KD 81.2±9.2mM) prior to the irreversible reaction, with maximum rate constants of 0.18±0.01min(-1). Protection from inactivation was afforded by substrate analogues demonstrating the specificity of the reaction. Structural analysis suggested that the modified residue is Arg111, which was confirmed by protein chemistry experiments. Site-directed mutagenesis was used in dissecting the role of Arg111 in substrate binding, specificity and catalytic mechanism. The mutant Arg111Ala enzyme exhibited unchanged Km value for GSH but showed reduced affinity for the xenobiotic substrates, higher kcat and specific activities towards aromatic substrates and lower specific activities towards aliphatic substrates. The biological significance of the specific modification of Arg111 by dicarbonyl compounds and the role of Arg111 as a target for engineering xenobiotic substrate specificity were discussed.

Biotherapeutic potential and mechanisms of action of colchicine.

Cancer is a clinical situation caused by uncontrolled cell division and is responsible for a large number of deaths worldwide. Colchicine is a classical antimitotic, tubulin-binding agent (TBA) which is being explored for its antitumor activities, although its tubulin-binding ability leads to some toxicity toward normal cells proliferation. Colchicine derivatives are considered as potent antitumor compounds with less toxicity compared to colchicine. Derivatives with substituted functional groups at A-ring (methoxy), B-ring (acetamide) or C-ring (methoxy) have been synthesized via chemical and microbial routes and show modified bioactivities and altered tropolonic functionality. Earlier reports, in combination with our group's research findings, suggest that microbial biotransformation is an efficient choice for the production of bioactive colchicine derivatives. This route has gained significant interest in the mass production of regio-specific, cost-effective, safe and eco-friendly derivatives. The present review paper critically analyzes and discusses the development and application of colchicine derivatives as a potent antitumor molecule and their production through a microbial transformation process. The information provided in this review might assist in the stimulation of new ideas regarding the development of alternative therapeutic agent(s) for cancer treatment.

Directed evolution of glutathione transferases towards a selective glutathione-binding site and improved oxidative stability.

BACKGROUND: Glutathione transferases (GSTs) are a family of detoxification enzymes that catalyze the conjugation of glutathione (GSH) to electrophilic compounds. METHODS: A library of alpha class GSTs was constructed by DNA shuffling using the DNA encoding the human glutathione transferase A1-1 (hGSTA1-1) and the rat glutathione transferase A1-1 (rGSTA1-1). RESULTS: Activity screening of the library allowed the selection of a chimeric enzyme variant (GSTD4) that displayed high affinity towards GSH and GSH-Sepharose affinity adsorbent, higher kcat/Km and improved thermal stability, compared to the parent enzymes. The crystal structures of the GSTD4 enzyme in free form and in complex with GSH were determined to 1.6Å and 2.3Å resolution, respectively. Analysis of the GSTD4 structure showed subtle conformational changes in the GSH-binding site and in electron-sharing network that may contribute to the increased GSH affinity. The shuffled variant GSTD4 was further optimized for improved oxidative stability employing site-saturation mutagenesis. The Cys112Ser mutation confers optimal oxidative stability and kinetic properties in the GSTD4 enzyme. CONCLUSIONS: DNA shuffling allowed the creation of a chimeric enzyme variant with improved properties, compared to the parent enzymes. X-ray crystallography shed light on how recombination of a specific segment from homologous GSTA1-1 together with point mutations gives rise to a new functionally competent enzyme with improved binding, catalytic properties and stability. GENERAL SIGNIFICANCE: Such an engineered GST would be useful in biotechnology as affinity tool in affinity chromatography as well as a biocatalytic matrix for the construction of biochips or enzyme biosensors.

Preserving enzymatic activity and enhancing biochemical stability of glutathione transferase by soluble additives under free and tethered conditions.

In the present study, we report the effect of four different soluble additives (sucrose, lactitol, superfloc c577, and dextran sulfate) on the stability of glutathione transferase 1 enzyme from Zea mays (ZmGSTF1-1) under free and tethered conditions at 4 and 25 °C. Among all additives, the best stabilizing effects were observed in the case of superfloc c577 and sucrose at both tested temperatures, yet at distinct concentrations at each condition. Those two stabilizing agents were further combined and potential positive synergistic effects were investigated. In addition, we assessed the long-term storage and operational stability of ZmGSTF1-1 under tethered conditions in the presence of additives, which provided the most conducive effects on its stability under free conditions. Our results strongly suggest that the presence of additives may be beneficial to the stability of the enzyme under both free and tethered conditions. Thermodynamic analysis of the free enzyme in the presence of sucrose, which exhibited the best stabilizing effect at both temperatures, shed light on the possible mechanism of action. Given the considerable importance of the development of GST-based biosensors with prolonged stability, the present work may be of general interest to researchers in the field of applied enzymology.