Spectroscopic analysis of the bacterially expressed head domain of rotavirus VP6.

The rotavirus capsid protein VP6 forms the middle of three protein layers and is responsible for many critical steps in the viral life cycle. VP6 as a structural protein can be used in various applications including as a subunit vaccine component. The head domain of VP6 (VP6H) contains key sequences that allow the protein to trimerize and that represent epitopes that are recognized by human antibodies in the viral particle. The domain is rich in ß-sheet secondary structures. Here, VP6H was solubilised from bacterial inclusion bodies and purified using a single affinity chromatography step. Spectral (far-UV circular dichroism and intrinsic tryptophan fluorescence) analysis revealed that the purified domain had native-like secondary and tertiary structures. The domain could maintain structure up to 44°C during thermal denaturation following which structural changes result in an intermediate forming and finally irreversible aggregation and denaturation. The chemical denaturation with urea and guanidinium hydrochloride produces intermediates that represent a loss in the cooperativity. The VP6H domain is stable and can fold to produce its native structure in the absence of the VP6 base domain but cannot be defined as an independent folding unit.

In Vitro α-Glucosidase and α-Amylase Inhibition, Cytotoxicity and Free Radical Scavenging Profiling of the 6-Halogeno and Mixed 6,8-Dihalogenated 2-Aryl-4-methyl-1,2-dihydroquinazoline 3-Oxides.

Series of the 6-bromo/iodo substituted 2-aryl-4-methyl-1,2-dihydroquinazoline-3-oxides and their mixed 6,8-dihalogenated (Br/I and I/Br) derivatives were evaluated for inhibitory properties against α-glucosidase and/or α-amylase activities and for cytotoxicity against breast (MCF-7) and lung (A549) cancer cell lines. The 6-bromo-2-phenyl substituted 3a and its corresponding 6-bromo-8-iodo-2-phenyl-substituted derivative 3i exhibited dual activity against α-glucosidase (IC50 = 1.08 ± 0.02 µM and 1.01 ± 0.05 µM, respectively) and α-amylase (IC50 = 5.33 ± 0.01 µM and 1.18 ± 0.06 µM, respectively) compared to acarbose (IC50 = 4.40 ± 0.05 µM and 2.92 ± 0.02 µM, respectively). The 6-iodo-2-(4-fluorophenyl)-substituted derivative 3f, on the other hand, exhibited strong activity against α-amylase and significant inhibitory effect against α-glucosidase with IC50 values of 0.64 ± 0.01 µM and 9.27 ± 0.02 µM, respectively. Compounds 3c, 3l and 3p exhibited the highest activity against α-glucosidase with IC50 values of 1.04 ± 0.03, 0.92 ± 0.01 and 0.78 ± 0.05 µM, respectively. Moderate cytotoxicity against the MCF-7 and A549 cell lines was observed for these compounds compared to the anticancer drugs doxorubicin (IC50 = 0.25 ± 0.05 µM and 0.36 ± 0.07 µM, respectively) and gefitinib (IC50 = 0.19 ± 0.04 µM and 0.25 ± 0.03 µM, respectively), and their IC50 values are in the range of 10.38 ± 0.08-25.48 ± 0.08 µM and 11.39 ± 0.12-20.00 ± 0.05 µM, respectively. The test compounds generally exhibited moderate to strong antioxidant capabilities, as demonstrated via robust free radical scavenging activity assays, viz., DPPH and NO. The potential of selected derivatives to inhibit superoxide dismutase (SOD) was also investigated via enzymatic assay in vitro. Molecular docking revealed the N-O moiety as essential to facilitate electrostatic interactions of the test compounds with the protein residues in the active site of α-glucosidase and α-amylase. The presence of bromine and/or iodine atoms resulted in increased hydrophobic (alkyl and/or π-alkyl) interactions and therefore increased inhibitory effect against both enzymes.

In Vitro Enzymatic and Kinetic Studies, and In Silico Drug-Receptor Interactions, and Drug-Like Profiling of the 5-Styrylbenzamide Derivatives as Potential Cholinesterase and ß-Secretase Inhibitors with Antioxidant Properties.

The 5-(styryl)anthranilamides were transformed into the corresponding 5-styryl-2-(p-tolylsulfonamido)benzamide derivatives. These 5-styrylbenzamide derivatives were evaluated through enzymatic assays in vitro for their capability to inhibit acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and ß-secretase (BACE-1) activities as well as for antioxidant potential. An in vitro cell-based antioxidant activity assay involving lipopolysaccharides (LPS)-induced reactive oxygen species (ROS) production revealed that compounds 2a and 3b have the capability of scavenging free radicals. The potential of the most active compound, 5-styrylbenzamide (2a), to bind copper (II) or zinc (II) ions has also been evaluated spectrophotometrically. Kinetic studies of the most active derivatives from each series against the AChE, BChE, and ß-secretase activities have been performed. The experimental results are complemented with molecular docking studies into the active sites of these enzymes to predict the hypothetical protein-ligand binding modes. Their drug likeness properties have also been predicted.

Bluetongue virus viral protein 7 stability in the presence of glycerol and sodium chloride.

PURPOSE: The Orbivirus Bluetongue virus (BTV) is an economically significant disease that affects mainly wild and domestic ruminants. BTV is most often seen symptomatically in sheep, but is easily carried by goats, cattle, and wild ruminants. To date there are several problems with the vaccines currently available for BTV, and one of the most promising candidates to increase vaccine efficacy is a protein-based vaccine, for which viral protein 7 (VP7) is a great candidate to be included in it. In order to further these studies, the stability of BTV VP7 in common vaccine additives needs to be investigated. MATERIALS AND METHODS: Recombinant BTV VP7 was expressed in a bacterial cell system and purified before being analysed using spectroscopic techniques including far-ultraviolet (UV) circular dichroism and intrinsic tryptophan fluorescence. BTV was analysed in a number of different buffer conditions. RESULTS: We report here that BTV VP7 maintains its native secondary structure until at least 52℃ and native-like tertiary structure to at least 80℃. Far-UV circular dichroism and intrinsic tryptophan fluorescence emission spectra indicate significant secondary and tertiary structure remaining even at 90℃, respectively. Six M guanidinium chloride is able to unfold BTV VP7 while 8 M urea could not. CONCLUSION: Twenty percent glycerol and 300 mM sodium chloride appear to have a protective effect on BTV VP7's structure, as significantly more structure is seen at 90℃ when compared to BTV VP7 without the addition of these chemicals. Both glycerol and sodium chloride are common vaccine additives.

Synthesis of furocoumarin-stilbene hybrids as potential multifunctional drugs against multiple biochemical targets associated with Alzheimer's disease.

A series of furocoumarin-stilbene hybrids has been synthesized and evaluated in vitro for inhibitory effect against acetylcholinesterase (AChE), butyrylcholinestarase (BChE), ß-secretase, cyclooxygenase-2 (COX-2), and lipoxygenase-5 (LOX-5) activities including free radical-scavenging properties. Among these hybrids, 8-(3,5-dimethoxyphenyl)-4-(3,5-dimethoxystyryl)furochromen-2-one 4h exhibited significant anticholinesterase activity and inhibitory effect against ß-secretase, COX-2 and LOX-5 activities. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and an in vitro cell-based antioxidant activity assay involving lipopolysaccharide induced reactive oxygen species production revealed that 4h has capability of scavenging free radicals. Molecular docking into AChE, BChE, ß-secretase, COX-2 and LOX-5 active sites has also been performed.

Benzofuran-selenadiazole hybrids as novel α-glucosidase and cyclooxygenase-2 inhibitors with antioxidant and cytotoxic properties.

Series of 2-arylbenzofuran-1,2,3-selenodiazole hybrids were prepared via multiple reactions and then evaluated in vitro through enzymatic assay for inhibitory effect against α-glucosidase and cyclooxygenase-2 (COX-2) activities including antioxidant activity. The presence of 1,2,3-selenodiazole moiety resulted in increased inhibitory effect for compounds 4a-f against α-glucosidase and COX-2 activities, and increased free radical scavenging activity. 6-Acetoxy-2-phenyl-5-(1,2,3-selenadiazol-4-yl)benzofuran (4a) and its 2-(4-methoxyphenyl) substituted derivative (4f) were, in turn, screened for antiproliferation against the breast MCF-7 cancer cell line and for cytotoxicity on the human embryonic kidney derived Hek293-T cells. A cell-based antioxidant activity assay involving lipopolysaccharide induced reactive oxygen species production in these cells was performed. Molecular docking has also been performed on these two compounds to predict protein-ligand interactions against α-glucosidase and COX-2.

Expanding our understanding of the role polyprotein conformation plays in the coronavirus life cycle.

Coronavirus are the causative agents in many globally concerning respiratory disease outbreaks such as severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS) and coronavirus disease-2019 (COVID-19). It is therefore important that we improve our understanding of how the molecular components of the virus facilitate the viral life cycle. These details will allow for the design of effective interventions. Krichel and coauthors in their article in the Biochemical Journal provide molecular details of how the viral polyprotein (nsp7-10) produced from the positive single stranded RNA genome, is cleaved to form proteins that are part of the replication/transcription complex. The authors highlight the impact the polyprotein conformation has on the cleavage efficiency of the main protease (Mpro) and hence the order of release of non-structural proteins 7-10 (nsp7-10) of the SARS-CoV. Cleavage order is important in controlling viral processes and seems to have relevance in terms of the protein-protein complexes formed. The authors made use of mass spectrometry to advance our understanding of the mechanism by which coronaviruses control nsp 7, 8, 9 and 10 production in the virus life cycle.

Synthesis, In Vitro Evaluation and Molecular Docking of the 5-Acetyl-2-aryl-6-hydroxybenzo[b]furans against Multiple Targets Linked to Type 2 Diabetes.

The 5-acetyl-2-aryl-6-hydroxybenzo[b]furans 2a-h have been evaluated through in vitro enzymatic assay against targets which are linked to type 2 diabetes (T2D), namely, α-glucosidase, protein tyrosine phosphatase 1B (PTP1B) and ß-secretase. These compounds have also been evaluated for antioxidant activity using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free-radical scavenging method. The most active compounds against α-glucosidase and/or PTP1B, namely, 4-fluorophenyl 2c, 4-methoxyphenyl 2g and 3,5-dimethoxyphenyl substituted 2h derivatives were also evaluated for potential anti-inflammatory properties against cyclooxygenase-2 activity. The Lineweaver-Burk and Dixon plots were used to determine the type of inhibition on compounds 2c and 2h against α-glucosidase and PTP1B receptors. The interactions were investigated in modelled complexes against α-glucosidase and PTP1B via molecular docking.

Synthesis, α-glucosidase inhibition and antioxidant activity of the 7-carbo-substituted 5-bromo-3-methylindazoles.

Series of 7-aryl- (3a-f), 7-arylvinyl- (3g-k) and 7-(arylethynyl)-5-bromo-3-methylindazoles (4a-f) have been evaluated through enzymatic assay in vitro for inhibitory effect against α-glucosidase activity and for antioxidant potential through the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. Compounds 3a-k and 4a-f showed significant to moderate α-glucosidase inhibition with IC50 values in the range of 0.50-51.51 µM and 0.42-23.71 µM compared with acarbose drug (IC50 = 0.82 µM), respectively. 5-Bromo-3-methyl-7-phenyl-1H-indazole (3a), 5-bromo-3-methyl-7-styryl-1H-indazole (3h) and 5-bromo-3-methyl-7-styryl-1H-indazole (4a) exhibited moderate to significant antigrowth effect against the breast MCF-7 cancer cell line and reduced cytotoxicity against the human embryonic kidney derived Hek293-T cells when compared to doxorubicin as reference standard. Non-covalent (alkyl, π-alkyl and π-π T shaped), electrostatic (π-sulfur and/or π-anion) and hydrogen bonding interactions are predicted to increase interactions with protein residues, thereby enhancing the inhibitory effect of these compounds against α-glucosidase.

Functional screening of a soil metagenome for DNA endonucleases by acquired resistance to bacteriophage infection.

Endonucleases play a crucial role as reagents in laboratory research and diagnostics. Here, metagenomics was used to functionally screen a fosmid library for endonucleases. A fosmid library was constructed using metagenomic DNA isolated from soil sampled from the unique environment of the Kogelberg Nature Reserve in the Western Cape of South Africa. The principle of acquired immunity against phage infection was used to develop a plate-based screening technique for the isolation of restriction endonucleases from the library. Using next-generation sequencing and bioinformatics tools, sequence data were generated and analysed, revealing 113 novel open reading frames (ORFs) encoding putative endonuclease genes and ORFs of unknown identity and function. One endonuclease designated Endo52 was selected from the putative endonuclease ORFs and was recombinantly produced in Escherichia coli Rosetta™ (DE3) pLysS. Endo52 was purified by immobilised metal affinity chromatography and yielded 0.437 g per litre of cultivation volume. Its enzyme activity was monitored by cleaving lambda DNA and pUC19 plasmid as substrates, and it demonstrated non-specific endonuclease activity. In addition to endonuclease-like genes, the screen identified several unknown genes. These could present new phage resistance mechanisms and are an opportunity for future investigations.

In Vitro Evaluation and Docking Studies of 5-oxo-5H-furo[3,2-g]chromene-6-carbaldehyde Derivatives as Potential Anti-Alzheimer's Agents.

A series of novel 2-carbo-substituted 5-oxo-5H-furo[3,2-g]chromene-6-carbaldehydes and their 6-(4-trifluoromethyl)phenylhydrazono derivatives have been prepared and evaluated for biological activity against the human acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The most active compounds from each series were, in turn, evaluated against the following enzyme targets involved in Alzheimer's disease, ß-secretase (BACE-1) and lipoxygenase-15 (LOX-15), as well as for anti-oxidant potential. Based on the in vitro results of ChE and ß-secretase inhibition, the kinetic studies were conducted to determine the mode of inhibition by these compounds. 2-(4-Methoxyphenyl)-5-oxo-5H-furo[3,2-g]chromene-6-carbaldehyde (2f), which exhibited significant inhibitory effect against all these enzymes was also evaluated for activity against the human lipoxygenase-5 (LOX-5). The experimental results were complemented with molecular docking into the active sites of these enzymes. Compound 2f was also found to be cytotoxic against the breast cancer MCF-7 cell line.

Exploring Biological Activity of 4-Oxo-4H-furo[2,3-h]chromene Derivatives as Potential Multi-Target-Directed Ligands Inhibiting Cholinesterases, ß-Secretase, Cyclooxygenase-2, and Lipoxygenase-5/15.

A series of 5-oxo-5H-furo[3,2-g]chromene-6-carbaldehydes and their hydrazone derivatives were evaluated as potential multi-target-directed ligands in vitro against cholinesterases, ß-secretase, cyclooxygenase-2, and lipoxygenase-15 (LOX-15), as well as for free radical-scavenging activities. The most active compounds against LOX-15 were also evaluated for activity against the human lipoxygenase-5 (LOX-5). Kinetic studies against AChE, BChE, and ß-secretase (BACE-1) were performed on 2-(3-fluorophenyl)- (3b) and 2-(4-chlorophenyl)-6-[(4-trifluoromethylphenyl)hydrazonomethyl]furo[3,2-h]chromen-5-one (3e) complemented with molecular docking (in silico) to determine plausible protein-ligand interactions on a molecular level. The docking studies revealed hydrogen and/or halogen bonding interactions between the strong electron-withdrawing fluorine atoms of the trifluoromethyl group with several residues of the enzyme targets, which are probably responsible for the observed increased biological activity of these hydrazone derivatives. The two compounds were found to moderately inhibit COX-2 and lipoxygenases (LOX-5 and LOX-15). Compounds 3b and 3e were also evaluated for cytotoxicity against the breast cancer MCF-7 cell line and Hek293-T cells.

Inhibitory Effects of Novel 7-Substituted 6-iodo-3-O-Flavonol Glycosides against Cholinesterases and ß-secretase Activities, and Evaluation for Potential Antioxidant Properties.

A series of 7-halogeno- (X = F, Cl, Br) and 7-methoxy-substituted acetylated 6-iodo-3-O-flavonol glycosides were prepared, and evaluated for inhibitory effect in vitro against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities. 7-Bromo-2-(4-chlorophenyl)-6-iodo-4H-chromen-4-one-3-O-2,3,4,6-O-tetraacetyl-ß-d-glucopyranoside (2k) and 7-bromo-6-iodo-2-(4-methoxyphenyl)-4H-chromen-4-one-3-O-2,3,4,6-O-tetraacetyl-ß-d-glucopyranoside (2l) exhibited significant inhibitory effect against AChE activity when compared to the activity of the reference standard, donepezil. Compound 2k was found to be selective against AChE and to exhibit reduced inhibitory effect against BChE activity. 6-Iodo-7-methoxy-2-(4-methoxyphenyl)-4H-chromen-4-one-3-O-2,3,4,6-O-tetraacetyl-ß-d-glucopyranoside (2p) was found to exhibit increased activity against BChE, more so than the activity of donepezil. The most active compounds were also evaluated for inhibitory effect against ß-secretase activity and for potential radical scavenging activities. The experimental data were complemented with molecular docking (in silico) studies of the most active compounds into the active sites of these enzymes.

Synthesis and Evaluation of the 4-Substituted 2-Hydroxy-5-Iodochalcones and Their 7-Substituted 6-Iodoflavonol Derivatives for Inhibitory Effect on Cholinesterases and ß-Secretase.

A series of 2-aryl-3-hydroxy-6-iodo-4H-chromen-4-ones substituted at the 7-position with a halogen atom (X = F, Cl and Br) or methoxy group and their corresponding 4-substituted 2-hydroxy-5-iodochalcone precursors were evaluated in vitro for inhibitory effect against acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and ß-secretase (BACE1) activities. Although moderate inhibitory effect was observed for the chalcones against AChE, derivatives 2h, 2j and 2n exhibited significant inhibitory effect against BChE and BACE-1. The 2-aryl-7-fluoro-8-iodoflavonols 3b and 3c, on the other hand, exhibited increased activity and selectivity against AChE and reduced effect on BACE-1. The flavonols 3h, 3i, 3k, 3l and 3p exhibited moderate inhibitory effect against AChE, but significant inhibition against BChE. Compounds 2j and 3l exhibited non-competitive mode of inhibition against BACE-1. Molecular docking predicted strong interactions with the protein residues in the active site of BACE-1 implying these compounds bind with the substrate. Similarly docking studies predicted interaction of the most active compounds with both CAS and PAS of either AChE or BChE with mixed type of enzyme inhibition confirmed by kinetic studies.

Solubilisation and purification of recombinant bluetongue virus VP7 expressed in a bacterial system.

Bluetongue virus (BTV) is an Orbivirus that has a profound economic impact due to direct loss of livestock as well as movement bans in an attempt to prevent the spread of the disease to susceptible areas. BTV VP7, along with VP3, forms the inner capsid core of the virus where it acts as the barrier between the outer layer and the inner core housing the genetic material. Purification of BTV VP7 has proven to be problematic and expensive mainly due to its insolubility is several expression systems. To overcome this, in this paper we present a protocol for the solubilisation of BTV VP7 from inclusion bodies expressed in E.coli, and subsequent purification using nickel affinity chromatography. The purified protein was then characterised using native PAGE, far ultraviolet circular dichroism (far-UV CD) and intrinsic fluorescence and found to have both secondary and tertiary structure even in the presence of 5 M urea. Both tertiary and secondary structure was further shown to be to be maintained at least to 42 °C in 5 M urea.

Analysis of Conserved, Computationally Predicted Epitope Regions for VP5 and VP7 Across three Orbiviruses.

Orbiviruses are double-stranded RNA viruses that have profound economic and veterinary significance, 3 of the most important being African horse sickness virus (AHSV), bluetongue virus (BTV), and epizootic hemorrhagic disease virus (EHDV). Currently, vaccination and vector control are used as preventative measures; however, there are several problems with the current vaccines. Comparing viral amino acid sequences, we obtained an AHSV-BTV-EHDV consensus sequence for VP5 (viral protein 5) and for VP7 (viral protein 7) and generated homology models for these proteins. The structures and sequences were analyzed for amino acid sequence conservation, entropy, surface accessibility, and epitope propensity, to computationally determine whether consensus sequences still possess potential epitope regions. In total, 5 potential linear epitope regions on VP5 and 11 on VP7, as well as potential discontinuous B-cell epitopes, were identified and mapped onto the homology models created. Regions identified for VP5 and VP7 could be important in vaccine design against orbiviruses.

Synthesis, Cytotoxicity and Molecular Docking Studies of the 9-Substituted 5-Styryltetrazolo[1,5-c]quinazoline Derivatives.

In this paper, we describe the synthesis of the 5-styryltetrazolo[1,5-c]quinazolines substituted at the 9-position with a 4-fluorophenyl ring directly or via a conjugated π-spacer (C=C or C≡C bond) based on the 6-bromo-4-chloro-2-styrylquinazoline scaffold. The structures of the synthesized compounds were characterized based on a combination of ¹H-NMR, 13C-NMR, IR and high resolution mass spectral data as well as microanalyses. The tetrazoloquinazolines were evaluated for potential in vitro cytotoxicity against the human breast adenocarcinoma (MCF-7) and cervical cancer (HeLa) cells. The anti-proliferative assays demonstrated that the 9-bromo-5-styryltetrazolo[1,5-c]quinazoline 3a and 9-bromo-5-(4-fluorostyryl)tetrazolo[1,5-c]quinazoline 3b exhibit significant cytotoxicity against both cell lines. A carbon-based substituent at the 9-position resulted in complete loss of cytotoxicity against both cell lines except for the 5,9-bis((E)-4-fluorostyryl)tetrazolo[1,5-c]quinazoline 4e, which was found to exhibit comparable cytotoxicity to that of Melphalan (IC50 = 61 µM) against the MCF-7 cell line with IC50 value of 62 µM. Molecular docking against tubulin (PDB:1TUB) showed that compounds 3a, 3b and 4e bind to the tubulin heterodimer. Binding involves hydrogen bonding for 3a and 3b and halogen interactions for 4e.

A conserved interdomain interaction is a determinant of folding cooperativity in the GST fold.

The canonical glutathione transferase (GST) fold found in many monomeric and dimeric proteins consists of two domains that differ in structure and conformational dynamics. However, no evidence exists that the two domains unfold/fold independently at equilibrium, indicating the significance of interdomain interactions in governing cooperativity between domains. Bioinformatics analyses indicate the interdomain interface of the GST fold is large, predominantly hydrophobic with a high packing density explaining cooperative interdomain behavior. Structural alignments reveal a topologically conserved lock-and-key interaction across the domain interface in which a bulky hydrophobic residue ("key") protrudes from the surface of the N-domain and inserts into a pocket ("lock") in the C-domain. To better understand the molecular basis for the contribution of interdomain interactions toward cooperativity within the GST fold in the absence of any influence from quaternary interactions, studies were done with two monomeric GST proteins: Escherichia coli Grx2 (EcGrx2) and human CLIC1 (hCLIC1). Replacing the methionine "key" residue with alanine is structurally nondisruptive, whereas it significantly diminishes the folding cooperativity of both proteins. The loss in cooperativity between domains in the mutants is reflected by a change in the equilibrium folding mechanism from a wild-type two-state process to a three-state process, populating a stable folding intermediate.

Stability of the domain interface contributes towards the catalytic function at the H-site of class alpha glutathione transferase A1-1.

Cytosolic glutathione transferases (GSTs) are major detoxification enzymes in aerobes. Each subunit has two distinct domains and an active site consisting of a G-site for binding GSH and an H-site for an electrophilic substrate. While the active site is located at the domain interface, the role of the stability of this interface in the catalytic function of GSTs is poorly understood. Domain 1 of class alpha GSTs has a conserved tryptophan (Trp21) in helix 1 that forms a major interdomain contact with helices 6 and 8 in domain 2. Replacing Trp21 with an alanine is structurally non-disruptive but creates a cavity between helices 1, 6 and 8 thus reducing the packing density and van der Waals contacts at the domain interface. This results in destabilization of the protein and a marked reduction in catalytic activity. While functionality at the G-site is not adversely affected by the W21A mutation, the H-site becomes more accessible to solvent and less favorable for the electrophilic substrate 1-chloro-2,4-dinitrobenzene (CDNB). Not only does the mutation result in a reduction in the energy for stabilizing the transition state formed in the S(N)Ar reaction between the substrates GSH and CDNB, it also compromises the ability of the enzyme to form and stabilize a transition state analogue (Meisenheimer complex) formed between GSH and 1,3,5-trinitrobenzene (TNB). The study demonstrates that the stability of the domain-domain interface plays a role in mediating the catalytic functionality of the active site, particularly the H-site, of class alpha GSTs.

The role of a topologically conserved isoleucine in glutathione transferase structure, stability and function.

The common fold shared by members of the glutathione-transferase (GST) family has a topologically conserved isoleucine residue at the N-terminus of helix 3 which is involved in the packing of helix 3 against two beta-strands in domain 1. The role of the isoleucine residue in the structure, function and stability of GST was investigated by replacing the Ile71 residue in human GSTA1-1 by alanine or valine. The X-ray structures of the I71A and I71V mutants resolved at 1.75 and 2.51 A, respectively, revealed that the mutations do not alter the overall structure of the protein compared with the wild type. Urea-induced equilibrium unfolding studies using circular dichroism and tryptophan fluorescence suggest that the mutation of Ile71 to alanine or valine reduces the stability of the protein. A functional assay with 1-chloro-2,4-dinitrobenzene shows that the mutation does not significantly alter the function of the protein relative to the wild type. Overall, the results suggest that conservation of the topologically conserved Ile71 maintains the structural stability of the protein but does not play a significant role in catalysis and substrate binding.