Engineering glutathione S-transferase with a point mutation at conserved F136 residue increases the xenobiotic-metabolizing activity.

Glutathione S-transferases (GSTs) are multifunctional enzymes that play major roles in a wide range of biological processes, including cellular detoxification, biosynthesis, metabolism, and transport. The dynamic structural scaffold and diverse functional roles of GSTs make them important for enzyme engineering and for exploring novel biotechnological applications. The present study reported a significant gain-of-function activity in GST caused by a point mutation at the conserved F136 residue. The fluorescence quenching and kinetic data suggested that both binding affinity and catalytic efficiency of the mutant enzyme to the substrates 1-chloro-2,4-dinitrobenzene (CDNB), as well as the glutathione (GSH), is increased. Molecular docking showed that the mutation improves the binding interactions of the GSH with several binding-site residues. The simulation of molecular dynamics revealed that the mutant enzyme gained increased structural rigidity than the wild-type enzyme. The mutation also altered the residue interaction network (RIN) of the GSH-binding residues. These phenomena suggested that mutations led to conformational alterations and dominant differential motions in the enzyme that lead to increased rigidity and modifications in RIN. Collectively, engineering GST with a single point mutation at conserved F136 can significantly increase its xenobiotic activity by increasing the catalytic efficiency that may be exploited for biotechnological applications.

Designing a vaccine for fascioliasis using immunogenic 24 kDa mu-class glutathione s-transferase.

Fascioliasis, caused by the liver fluke Fasciola gigantica, is a significant zoonotic disease of the livestock and human, causing substantial economic loss worldwide. Triclabendazole (TCBZ) is the only drug available for the management of the disease against which there is an alarming increase in drug resistance. No vaccine is available commercially for the protection against this disease. Increasing resistance to TCBZ and the lack of a successful vaccine against fascioliasis demands the development of vaccines. In the present study, a structural immunoinformatics approach was used to design a multi-epitope subunit vaccine using the glutathione S-transferase (GST) protein of Fasciola gigantica. The GST antigen is a safe, non-allergic, highly antigenic, and effective vaccine candidate against various parasitic flukes and worms. The cytotoxic T lymphocytes, helper T lymphocytes, and B-cell epitopes were selected for constructing the vaccine based on their immunogenic behavior and binding affinity. The physicochemical properties, allergenicity, and antigenicity of the designed vaccine were analyzed. To elucidate the tertiary structure of the vaccine, homology modeling was performed, followed by structure refinement and docking against the TLR2 immune receptor. Molecular dynamics simulations showed a stable interaction between the vaccine and the receptor complex. Finally, in silico cloning was performed to evaluate the expression and translation of the vaccine construct in the E. coli expression system. Further studies require experimental validation for the safety and immunogenic behavior of the designed vaccine.

Draft Genome of the Liver Fluke Fasciola gigantica.

Fascioliasis, a neglected foodborne disease caused by liver flukes (genus Fasciola), affects more than 200 million people worldwide. Despite technological advances, little is known about the molecular biology and biochemistry of these flukes. We present the draft genome of Fasciola gigantica for the first time. The assembled draft genome has a size of ∼1.04 Gb with an N50 and N90 of 129 and 149 kb, respectively. A total of 20 858 genes were predicted. The de novo repeats identified in the draft genome were 46.85%. The pathway included all of the genes of glycolysis, Krebs cycle, and fatty acid metabolism but lacked the key genes of the fatty acid biosynthesis pathway. This indicates that the fatty acid required for survival of the fluke may be acquired from the host bile. It may be hypothesized that the relatively larger F. gigantica genome did not evolve through genome duplications but rather is interspersed with many repetitive elements. The genomic information will provide a comprehensive resource to facilitate the development of novel interventions for fascioliasis control.

Abnormal Microtubule Dynamics Impair the Nuclear-Cytoplasmic Transport in Dementia.

Disrupted nuclear-cytoplasmic transport (NCT) is a common pathophysiological event in several neurodegenerative disorders. However, the correlation between the mutations in the pathogenic microtubule-associated protein tau and NCT and neuronal dysfunction is not yet clearly understood. A recent study revealed that tau is mislocalized to the neuronal cell body and, thus, deforms the nuclear membrane in the frontotemporal dementia (FTD). This causes a defect in NCT, leading to neurodegeneration. The microtubule depolymerization could rescue the NCT defects as well as neurodegeneration. Therefore, agents that can modulate the microtubule functions or NCT can constitute a potential therapeutic method for the treatment of neurodegenerative disorders.

Structural basis of urea-induced unfolding of Fasciola gigantica glutathione S-transferase.

Glutathione S-transferases (GSTs) are enzymes that are involved in the detoxification of harmful electrophilic endogenous and exogenous compounds by conjugating with glutathione (GSH). The liver fluke GSTs have multifunctional roles in the host-parasite interaction, such as general detoxification and bile acid sequestration to synthase activity. The GSTs have been highlighted as vaccine candidates towards parasitic flukes. In this study, we have thoroughly examined the urea-induced unfolding of a mu-class Fasciola gigantica GST1 (FgGST1) using spectroscopic techniques and molecular dynamic simulations. FgGST1 is a highly cooperative molecule, because during urea-induced equilibrium unfolding, a concurrent unfolding of the protein without stabilization of any folded intermediate was observed. The protein was stabilized with conformational free energy of about ~12.36 kcal/mol. The protein loses its activity with increasing urea concentration, as the GSH molecule is not able to bind to the protein. We also studied the fluorescence quenching of Trp residues and the obtained K SV data that provided additional information on the unfolding of FgGST1. Molecular dynamic trajectories simulated in different urea concentrations and temperatures indicated that urea destabilizes FgGST1 structure by weakening hydrophobic interactions and the hydrogen bond network. We observed a precise correlation between the in vitro and in silico studies.

Unfolding of Acinetobacter baumannii MurA proceeds through a metastable intermediate: A combined spectroscopic and computational investigation.

Peptidoglycan (PG) is the main constituent of the bacterial cell wall. The enzyme UDP­N­acetylglucosamine enolpyruvyl transferase (MurA) catalyzes the transfer of enolpyruvate from phosphoenolpyruvate to uridinediphospho­N­acetylglucosamine, which is the first committed step of PG biosynthesis. In this study, we have systematically examined the urea-induced unfolding of Acinetobacter baumannii MurA (AbMurA) using various optical spectroscopic techniques and molecular dynamics (MD) simulations. The urea-induced unfolding of AbMurA was a three-state process, where a metastable intermediate conformation state is populated between 3.0 and 4.0 M. Above 6.0 M urea, AbMurA gets completely unfolded. The transition from the native structure to the partially unfolded metastable state involves ~30% loss of native contacts but little change in the radius of gyration or core hydration properties. The intermediate-to-unfolded state transition was characterized by a large increase in the radius of gyration. MD trajectories simulated in different unfolding conditions suggest that urea destabilizes AbMurA structure weakening hydrophobic interactions and the hydrogen bond network. We observed a clear correlation between both in vitro and in silico studies. To our knowledge, this is also the first report on unfolding/stability analysis of any MurA enzyme.

Comprehensive analysis of the catalytic and structural properties of a mu-class glutathione s-transferase from Fasciola gigantica.

Glutathione S‒transferases (GSTs) play an important role in the detoxification of xenobiotics. They catalyze the nucleophilic addition of glutathione (GSH) to nonpolar compounds, rendering the products water-soluble. In the present study, we investigated the catalytic and structural properties of a mu-class GST from Fasciola gigantica (FgGST1). The purified recombinant FgGST1 formed a homodimer composed of 25 kDa subunit. Kinetic analysis revealed that FgGST1 displays broad substrate specificity and shows high GSH conjugation activity toward 1-chloro-2,4-dinitrobenzene, 4-nitroquinoline-1-oxide, and trans-4-phenyl-3-butene-2-one and peroxidase activity towards trans-2-nonenal and hexa-2,4-dienal. The FgGST1 was highly sensitive to inhibition by cibacron blue. The cofactor (GSH) and inhibitor (cibacron blue) were docked, and binding sites were identified. The molecular dynamics studies and principal component analysis indicated the stability of the systems and the collective motions, respectively. Unfolding studies suggest that FgGST1 is a highly cooperative molecule because, during GdnHCl-induced denaturation, a simultaneous unfolding of the protein without stabilization of any partially folded intermediate is observed. The protein is stabilized with a conformational free energy of about 10 ± 0.3 kcal mol-1. Additionally, the presence of conserved Pro-53 and structural motifs such as N-capping box and hydrophobic staple, further aided in the stability and proper folding of FgGST1.

UDP-N-Acetylglucosamine enolpyruvyl transferase (MurA) of Acinetobacter baumannii (AbMurA): Structural and functional properties.

Peptidoglycan (PG) is the key component of the bacterial cell wall. The enzyme UDP-N-Acetylglucosamine Enolpyruvyl Transferase (MurA) catalyzes the transfer of enolpyruvate from phosphoenolpyruvate (PEP) to uridinediphospho-N-acetylglucosamine (UNAG), which is the first committed step of PG biosynthesis. Here, we present the biochemical and structural features of the MurA enzyme of the opportunistic pathogen Acinetobacter baumannii (AbMurA). The recombinant AbMurA exists as a monomer in solution and shows optimal activity at pH 7.5 and 37°C. The Km for UDP-N-acetylglucosamine was 1.062±0.09mM and for PEP was 1.806±0.23mM. The relative enzymatic activity was inhibited ∼3 fold in the presence of 50mM fosfomycin (FFQ). Superimposition of the AbMurA model with E. coli demonstrated key structural similarity in the FFQ-binding site. AbMurA also has a surface loop that contains the active site Cys116 that interact with FFQ. Sequence analysis indicates the presence of the five conserved amino acids, i.e., K22, C116, D306, D370 and L371, required for the functional activity like other MurA enzymes from different bacteria. MurA enzymes are indispensable for cell integrity and their lack of counterparts in eukaryotes suggests them to be a promising drug target.