Molecular and biochemical characterization of a plant-like iota-class glutathione S-transferase from the halotolerant cyanobacterium Halothece sp. PCC7418.

AIMS: This study identifies a unique glutathione S-transferase (GST) in extremophile using genome, phylogeny, bioinformatics, functional characterization, and RNA sequencing analysis. METHODS AND RESULTS: Five putative GSTs (H0647, H0729, H1478, H3557, and H3594) were identified in Halothece sp. PCC7418. Phylogenetic analysis suggested that H0647, H1478, H0729, H3557, and H3594 are distinct GST classes. Of these, H0729 was classified as an iota-class GST, encoding a high molecular mass GST protein with remarkable features. The protein secondary structure of H0729 revealed the presence of a glutaredoxin (Grx) Cys-Pro-Tyr-Cys (C-P-Y-C) motif that overlaps with the N-terminal domain and harbors a topology similar to the thioredoxin (Trx) fold. Interestingly, recombinant H0729 exhibited a high catalytic efficiency for both glutathione (GSH) and 1-chloro-2, 4-dinitrobenzene (CDNB), with catalytic efficiencies that were 155- and 32-fold higher, respectively compared to recombinant H3557. Lastly, the Halothece gene expression profiles suggested that antioxidant and phase II detoxification encoding genes are crucial in response to salt stress. CONCLUSION: Iota-class GST was identified in cyanobacteria. This GST exhibited a high catalytic efficiency toward xenobiotic substrates. Our findings shed light a diversified evolution of GST in cyanobacteria and provide functional dynamics of the genes encoding the enzymatic antioxidant and detoxification systems under abiotic stresses.

1H, 13C, 15N backbone resonance assignment of Escherichia coli adenylate kinase.

Adenylate kinase reversibly catalyzes the conversion of ATP plus AMP to two ADPs. This essential catalyst is present in every cell, and the Escherichia coli protein is often employed as a model enzyme. Our aim is to use the E. coli enzyme to understand dry protein structure and protection. Here, we report the expression, purification, steady-state assay, NMR conditions and 1H, 13C, 15N backbone resonance NMR assignments of its C77S variant. These data will also help others utilize this prototypical enzyme.

Structural Dynamics of the Precatalytic State of Human Cytochrome c upon T28C, G34C, and A50C Mutations: A Molecular Dynamics Simulation Perspective.

The native structure of cytochrome c (cytc) contains hexacoordinate heme iron with His18 and Met80 residues ligated at the axial sites. Mutations of cytc at Ω-loops have been investigated in modulating the peroxidase activity and, hence, related to the initiation of the apoptotic pathway. Our previous experimental data reported on the peroxidase activity of the cysteine-directed mutants at different parts of the Ω-loop of human cytc (hCytc), that is, T28C, G34C, and A50C. In this work, we performed 1 µs molecular dynamics (MD) simulations to elucidate the detailed structural and dynamic changes upon these mutations, particularly at the proximal Ω-loop. The structures of hCytc were modeled in the hexacoordinated form, which was referred to as the "precatalytic state". The results showed that the structural features of the G34C mutant were more distinctive than those of other mutants. G34C mutation caused local destabilization and flexibility at the proximal Ω-loop (residues 12-28) and an extended distance between this Ω-loop region and heme iron. Besides, analysis of the orientation of the Arg38 side chain of the G34C mutant revealed the Arg38 conformer facing away from the heme iron. The obtained MD results also suggested structural diversity of the precatalytic states for the three hCytc mutants, specifically the effect of G34C mutation on the flexibility of the proximal Ω-loops. Therefore, our MD simulations combined with previous experimental data provide detailed insights into the structural basis of hCytc that could contribute to its pro-apoptotic function.

Molecular insights on the conformational dynamics of a P76C mutant of human cytochrome c and the enhancement on its peroxidase activity.

In apoptotic pathway, the interaction of Cytochrome c (Cytc) with cardiolipin in vivo is a key process to induce peroxidase activity of Cytc and trigger the release of Cytc in the inner mitochondria into cytosol. The peroxidase active form of Cytc occurs due to local conformational changes that support the opening of the heme crevice and the loss of an axial ligand between Met80 and heme Fe. Structural adjustments at the Ω-loop segments of Cytc are required for such process. To study the role of the distal Ω-loop segments comprising residues 71-85 in human Cytc (hCytc), we investigated a cysteine mutation at Pro76, one of the highly conserved residues in this loop. The effect of P76C mutant was explored by the combination of experimental characterizations and molecular dynamics (MD) simulations. The peroxidase activity of the P76C mutant was found to be significantly increased by ∼13 folds relative to the wild type. Experimental data on global denaturation, alkaline transition, heme bleaching, and spin-labeling Electron Spin Resonance were in good agreement with the enhancement of peroxidase activity. The MD results of hCytc in the hexacoordinate form suggest the important changes in P76C mutant occurred due to the unfolding at the central Ω-loop (residues 40-57), and the weakening of H-bond between Tyr67 and Met80. Whereas the experimental data implied that the P76C mutant tend to be in equilibrium between the pentacoordinate and hexacoordinate forms, the MD and experimental information are complementary and were used to support the mechanisms of peroxidase active form of hCytc.

Influence of cysteine-directed mutations at the Ω-loops on peroxidase activity of human cytochrome c.

Cytochrome c (Cytc) is a multifunctional protein associated with electron shuttling in the inner membrane of mitochondria and also involving in the apoptotic pathway. It has been identified that mutations located in the flexible central 40-57 Ω-loop including the naturally occurring G41S, Y48H, and A51V mutants, which are found in patients with thrombocytopenia 4, a platelet disorder, alter the structural properties of human Cytc (hCytc) that associated to enhanced peroxidase activity. In this work we compared the cysteine-directed mutants of hCytc located in three different parts of Ω-loops, i.e., T28C and G34C (proximal Ω-loop), and A50C (central Ω-loop), with respect to the wild-type (WT) hCytc. The mutants and WT hCytc were structurally characterized by circular dichroism, heating and chemical denaturations, and fluorescence spectroscopy. The flexibility at the cysteine mutated sites was directly determined by site-directed spin-labeling Electron Spin Resonance. Alkaline transitions were determined by pH titration and the alkaline conformers were related to peroxidase activity of all hCytc proteins. Structural and dynamic characterizations were rationally correlated to the modulation of peroxidase activity in these mutants in comparison to the WT hCytc. We found that the cysteine mutations at residues T28 and G34, both located in the same region of Ω-loop, developed different conformations and dynamical properties that lead to different effects on the rates of peroxidase activity (G34C was ~2.6 folds higher), whereas the rate of G34C was closer to that of A50C mutant. The results implied that the flexibility and local structures of the proximal Ω-loop could also play an important role in modulating the peroxidase activity which can be associated to apoptosis.