Guanidine hydrochloride- and urea-induced unfolding of Toxoplasma gondii ferredoxin-NADP<SUP>+</SUP> reductase: stabilization of a functionally inactive holo-intermediate

Usually during the folding/unfolding of flavoproteins, an apo-intermediate is stabilized before global unfolding of the enzymes occurs. However, stabilization of a holo-intermediate has also been reported for a few flavoproteins. We have studied the unfolding of Toxoplasma gondii ferredoxin-NADP+ reductase (TgFNR) using GdnHCl and urea. A functionally inactive holo-intermediate of the enzyme was found to be stabilized during this unfolding process. The intermediate species had cofactor FAD bound to it, but it showed free movement due to which the stabilized intermediates were functionally inactive. The native TgFNR behaves cooperatively with the two structural domains interacting strongly with each other. The denaturants GdnHCl and urea, at low concentrations, were found to interact selectively with the NADP+-binding domain of TgFNR and to induce structural modifications in it. These selective modifications in the protein molecule lead to loss of interactions between two domains and the enzyme behaved non-cooperatively resulting in stabilization of an intermediate species. Significant differences in the structural properties of the GdnHCl- and urea-stabilized holo-intermediates of TgFNR were observed. Comparison of the unfolding pathway of TgFNR (a plant-type FNR) with that of FprA (a GR-type FNR) demonstrates that they follow very different pathways of unfolding

Cation induced differential effect on structural and functional properties of Mycobacterium tuberculosis α-Isopropylmalate synthase

<p>Abstract</p> <p>Background</p> <p>α-isopropylmalate synthase (MtαIPMS), an enzyme that catalyzes the first committed step of the leucine biosynthetic pathway of <it>Mycobacterium tuberculosis </it>is a potential drug target for the anti-tuberculosis drugs. Cations induce differential effect of activation and inhibition of MtαIPMS. To date no concrete mechanism for such an opposite effect of similarly charged cations on the functional activity of enzyme has been presented.</p> <p>Results</p> <p>Effect of cations on the structure and function of the MtαIPMS has been studied in detail. The studies for the first time demonstrate that different cations interact specifically at different sites in the enzyme and modulate the enzyme structure differentially. The inhibitors Zn²⁺and Cd²⁺ions interact directly with the catalytic domain of the enzyme and induce unfolding/denaturation of the domain. The activator K⁺also interacts with the catalytic TIM barrel domain however, it does not induce any significant effect on the enzyme structure. Studies with isolated catalytic TIM barrel domain showed that it can carry out the catalytic function on its own but probably requires the non-catalytic C-terminal domain for optimum functioning. An important observation was that divalent cations induce significant interaction between the regulatory and the catalytic domain of MtαIPMS thus inducing structural cooperativity in the enzyme. This divalent cation induced structural cooperativity might result in modulation of activity of the catalytic domain by regulatory domain.</p> <p>Conclusion</p> <p>The studies for the first time demonstrate that different cations bind at different sites in the enzyme leading to their differential effects on the structure and functional activity of the enzyme.</p

Unusual structural, functional, and stability properties of serine hydroxymethyltransferase from Mycobacterium tuberculosis

From the genome analysis of the Mycobacterium tuberculosis two putative genes namely GlyA and GlyA2 have been proposed to encode for the enzyme serine hydroxymethyltransferase. We have cloned, overexpressed, and purified to homogeneity their respective protein products, serine hydroxymethyltransferase, SHM1 and SHM2. The recombinant SHM1 and SHM2 exist as homodimers of molecular mass about 90 kDa under physiological conditions, however, SHM2 has more compact conformation and higher thermal stability than SHM1. The most interesting structural observation was that the SHM1 contains 1 mol of pyridoxal 5&#8242;-phosphate (PLP)/mol of enzyme dimer. This is the first report of such a unique stoichiometry of PLP and enzyme dimer for SHMT. The SHM2 contains 2 mol of PLP/mol of enzyme dimer, which is the usual stoichiometry reported for SHMT. Functionally both the recombinant enzymes showed catalysis of reversible interconversion of serine and glycine and aldol cleavage of a 3-hydroxyamino acid. However, unlike SHMT from other sources both SHM1 and SHM2 do not undergo half-transamination reaction with d-alanine resulting in formation of apoenzyme but l-cysteine removed the prosthetic group, PLP, from both the recombinant enzymes leaving the respective inactive apoenzymes. Comparative structural studies on the two enzymes showed that the SHM1 is resistant to alkaline denaturation up to pH 10.5, whereas the native SHM2 dimer dissociates into monomer at pH 9. urea- and guanidinium chloride-induced two-step unfolding of SHM1 and SHM2 with the first step being dissociation of dimer into apomonomer at low denaturant concentrations followed by unfolding of the stabilized monomer at higher denaturant concentrations

Self-assembly of bacteriophage-associated hyaluronate lyase (HYLP2) into an enzymatically active fibrillar film

The in vitro assembly of a soluble protein into its mature fibrillar form is usually accompanied by loss of its functional activity. Our study is the first demonstration of a natural enzyme (HylP2) retaining its enzymatic activity on conversion from pre-fibril to mature fibril and supports the contention that minor conformational changes in the native folded form of a protein can lead to the formation of a functional fibril. Hyaluronate lyase (HylP2) is a natural enzyme of bacteriophage 10403 of Streptococcus pyogenes. At pH 5.0, the enzyme undergoes partial unfolding localized in its N-terminal domain while the C-terminal domain maintains its folded trimeric conformation. This structural variant of HylP2 retains about 70% enzymatic activity with hyaluronan. It further self-assembles into a fibrillar film in vitro through solvent-exposed nonpolar surfaces and intermolecular &#946;-sheet formation by the &#946;-strands in the protein. Interestingly, the mature fibrillar film of HylP2 also retains about 60 and 20% enzymatic activity for hyaluronic acid and chondroitin sulfate, respectively. The possession of broad substrate specificity by the fibrillar form of HylP2 indicates that fluctuations in pH, which do not lead to loss of functionality of HylP2, might assist in bacterial pathogenesis. The formation of fibrillar film-like structure has been observed for the first time among the hyaluronidase enzymes. After acquiring this film-like structure in bacteriophage, HylP2 still retains its enzymatic activity, which establishes that these fibrils are a genuinely acquired protein fold/structure

Characterization of pyridoxal 5'-phosphate-binding domain and folding intermediate of Bacillus subtilis serine hydroxymethyltransferase: an autonomous folding domain

The pyridoxal-5'-phosphate-binding domain (PLPbd) of bsSHMT (Bacillus subtilis serine hydroxymethyltransferase) was cloned and over-expressed in Escherichia coli. The recombinant protein was solublized, refolded and purified from inclusion bodies by rapid mixing followed by ion exchange chromatography. Structural and functional studies suggested the native form of the domain, which obtained as a monomer and had similar secondary and tertiary structural properties as when present in the bsSHMT. The domain also binds to the PLP however with slightly lesser affinity than the native enzyme. GdmCl (guanidium chloride)-induced equilibrium unfolding of the recombinant PLP-binding domain showed a single monophasic transition which corresponds with the second phase transition of the GdmCl-induced unfolding of bsSHMT. The results indicate that PLPbd of bsSHMT is an independent domain, which attains its tertiary structure before the dimerization of partially folded monomer and behaves as a single cooperative unfolding unit under equilibrium conditions

Streptococcus pneumoniae hyaluronate lyase contains two non-cooperative independent folding/unfolding structural domains

Hyaluronate lyase contributes directly to bacterial invasion by degrading hyaluronan, the major component of host extracellular matrix of connective tissues. Streptococcus pneumoniae hyaluronate lyase (SpnHL) is built from two structural domains that interact through interface residues, in addition to being connected by a peptide linker. For the first time we demonstrate that the N- and C-terminal domains of SpnHL fold/unfold independent of each other suggesting the absence of any significant cooperative interactions between them. The C-terminal domain of SpnHL is less stable than the N-terminal domain against thermal and guanidine hydrochloride denaturation. The intact n-terminal domain was purified after limited proteolysis of SpnHL under conditions where only the C-terminal domain was unfolded. Isolated N-terminal domain of SpnHL had similar thermal stability as when present in the native enzyme and was found to be enzymatically active demonstrating that it is capable of carrying out enzymatic reaction on its own. Functional studies demonstrated that guanidine hydrochloride, guanidine isothiocyanate, l-arginine methyl ester, and l-arginine inhibit the enzymatic activity of SpnHL at very low concentrations. This provides a lead for new chemical entities that can be exploited for designing effective inhibitors of SpnHL

Ionic-strength-dependent transition of hen egg-white lysozyme at low pH to a compact state and its aggregation on thermal denaturation

Equilibrium acid-induced unfolding of hen egg-white lysozyme has been investigated by a combination of optical methods, size-exclusion chromatography, and differential scanning calorimetry. The results showed the presence of a partially folded state of hen egg-white lysozyme at pH 1.5, characterized by a substantial secondary structure, a large solvent exposure of non-polar clusters, and significantly disrupted tertiary structure. A large enthalpy was also associated with the conversion of the acid-unfolded state to a fully unfolded state. Size-exclusion chromatography and 8-anilino-1-naphthalenesulphonic acid-binding studies showed an ionic-strength-induced transition of the partially folded state to a compact conformation. Furthermore, an ionic-strength-dependent aggregation on thermal unfolding of the partially folded intermediate was also observed. These observations provide insights into the possible features responsible for the stabilization of intermediates in the folding of hen egg-white lysozyme

Glutathione mediated regulation of oligomeric structure and functional activity of Plasmodium falciparum glutathione S-transferase

<p>Abstract</p> <p>Background</p> <p>In contrast to many other organisms, the malarial parasite <it>Plasmodium falciparum </it>possesses only one typical glutathione <it>S</it>-transferase. This enzyme, <it>Pf</it>GST, cannot be assigned to any of the known GST classes and represents a most interesting target for antimalarial drug development. The <it>Pf</it>GST under native conditions forms non-covalently linked higher aggregates with major population (~98%) being tetramer. However, in the presence of 2 mM GSH, a dimer of <it>Pf</it>GST is observed. Recently reported study on binding and catalytic properties of <it>Pf</it>GST indicated a GSH dependent low-high affinity transition with simultaneous binding of two GSH molecules to <it>Pf</it>GST dimer suggesting that GSH binds to low affinity inactive enzyme dimer converting it to high affinity functionally active dimer. In order to understand the role of GSH in tetramer-dimer transition of <it>Pf</it>GST as well as in modulation of functional activity of the enzyme, detailed structural, functional and stability studies on recombinant <it>Pf</it>GST in the presence and absence of GSH were carried out.</p> <p>Results</p> <p>Our data indicate that the dimer – and not the tetramer – is the active form of <it>Pf</it>GST, and that substrate saturation is directly paralleled by dissociation of the tetramer. Furthermore, this dissociation is a reversible process indicating that the tetramer-dimer equilibrium of <it>Pf</it>GST is defined by the surrounding GSH concentration. Equilibrium denaturation studies show that the <it>Pf</it>GST tetramer has significantly higher stability compared to the dimer. The enhanced stability of the tetramer is likely to be due to stronger ionic interactions existing in it.</p> <p>Conclusion</p> <p>This is the first report for any GST where an alteration in oligomeric structure and not just small conformational change is observed upon GSH binding to the enzyme. Furthermore we also demonstrate a reversible mechanism of regulation of functional activity of <it>Plasmodium falciparum </it>glutathione <it>S</it>-transferase via GSH induced dissociation of functionally inactive tetramer into active dimers.</p

Entamoeba histolytica Phosphoserine aminotransferase (EhPSAT): insights into the structure-function relationship

<p>Abstract</p> <p>Background</p> <p>Presence of phosphorylated Serine biosynthesis pathway upstream to the de novo cysteine biosynthesis pathway makes PSAT a crucial enzyme. Besides this, phoshoserine produced by the enzyme can also be taken up directly by cysteine synthase as a substrate. PSAT is a PLP dependent enzyme where the cofactor serves as an epicenter for functional catalysis with the active site architecture playing crucial role in optimum function of the enzyme.</p> <p>Findings</p> <p>EhPSAT is a homodimer of molecular mass 86 kDa. To understand the structural modulations associated with pH dependent changes in functional activity of EhPSAT detailed biophysical studies were carried out. pH alterations had no significant effect on the secondary structure, cofactor orientation and oligomeric configuration of the enzyme however, pH dependent compaction in molecular dimensions was observed. Most interestingly, a direct correlation between pH induced modulation of functional activity and orientation of Trp 101 present in the active site of the enzyme was observed. Sodium halides nullified the pH induced global changes in the enzyme, however differential effect of these salts on the active site microenvironment and functional activity of the enzyme was observed.</p> <p>Conclusions</p> <p>The study unequivocally demonstrates that pH induced selective modification of active site microenvironment and not global change in structure or oligomeric status of the enzyme is responsible for the pH dependent change in enzymatic activity of PSAT.</p

Alkaline unfolding and salt-induced folding of bovine liver catalase at high pH

We have studied the alkaline unfolding of bovine liver catalase and its dependence on ionic strength by enzymic activity measurements and a combination of optical methods like circular dichroism, fluorescence and absorption spectroscopies. Under conditions of high pH (11.5) and low ionic strength, the native tetrameric enzyme dissociates into monomers with complete loss of enzymic activity and a significant loss of &#945;-helical content. Increase in ionic strength by addition of salts like potassium chloride and sodium sulphate resulted in folding of alkaline-unfolded enzyme by association of monomers to tetramer but with significantly different structural properties compared to native enzyme. The salt-induced tetrameric intermediate is characterized by a significant exposure of the buried hydrophobic clusters and significantly reduced &#945;-helical content compared to the native enzyme. The refolding/reconstitution studies showed that the salt-induced partially folded tetrameric intermediate shows significantly higher efficiency of refolding/reconstitution as compared to alkaline-denatured catalase in the absence of salts. These studies suggest that folding of multimeric enzymes proceeds probably through the hydrophobic collapse of partially folded multimeric intermediate with exposed hydrophobic clusters