Studies on active site mutants of P-falciparum adenylosuccinate synthetase: Insights into enzyme catalysis and activation

Adenylosuccinate synthetase catalyzes a reversible reaction utilizing IMP, GTP and aspartate in the presence of Mg2+ to form adenylosuccinate, GDP and inorganic phosphate. Comparison of similarly liganded complexes of Plasmodium falciparum, mouse and Escherichia coil AdSS reveals H-bonding interactions involving nonconserved catalytic loop residues (Asn429, Lys62 and Thr307) that are unique to the parasite enzyme. Site-directed mutagenesis has been used to examine the role of these interactions in catalysis and structural organization of P. falciparum adenylosuccinate synthetase (PfAdSS). Mutation of Asn429 to Val, Lys62 to Leu and Thr307 to Val resulted in an increase in K-m values for IMP, GTP and aspartate, respectively along with a 5 fold drop in the k(cat) value for N429V mutant suggesting the role of these residues in ligand binding and/or catalysis. We have earlier shown that the glycolytic intermediate, fructose 1,6 bisphosphate, which is an inhibitor of mammalian AdSS is an activator of the parasite enzyme. Enzyme kinetics along with molecular docking suggests a mechanism for activation wherein F16BP seems to be binding to the Asp loop and inducing a conformation that facilitates aspartate binding to the enzyme active site. Like in other AdSS, a conserved arginine residue (Arg155) is involved in dimer crosstalk and interacts with IMP in the active site of the symmetry related subunit of PfAdSS. We also report on the iochemical characterization of the arginine mutants (R155L, R155K and R155A) which suggests that unlike in E. coil AdSS, Arg155 in PfAdSS influences both ligand binding and catalysis. (C) 2010 Elsevier B.V. All rights reserved.

The Blackett Memorial Lecture, 1991: Chemical Insights into High-Temperature Superconductors

The high-temperature superconductors are complex oxides, generally containing two-dimensional CuO2 sheets. Various families of the cuprate superconductors are described, paying special attention to aspects related to oxygen stoichiometry, phase stability, synthesis and chemical manipulation of charge carriers. Other aspects discussed are chemical applications of cuprates, possibly as gas sensors and copper-free oxide superconductors. All but the substituted Nd and Pr cuprates are hole-superconductors. Several families of cuprates show a nearly constant n(h) at maximum T(c). Besides this universality, the cuprates exhibit a number of striking common features. Based on Cu(2p) photoemission studies, it is found that the Cu-O charge-transfer energy, DELTA, and the Cu(3d)-O(2p) hybridization strength, t(pd), are key factors in the superconductivity of cuprates. The relative intensity of the satellite in the Cu(2p) core-level spectra, the polarizability of the CuO2 sheets as well as the hole concentration are related to DELTA/t(pd). These chemical bonding factors have to be explicitly taken into account in any model for superconductivity of the cuprates.

Noble metal ionic sites for catalytic hydrogen combustion: spectroscopic insights

A catalytic hydrogen combustion reaction was carried out over noble metal catalysts substituted in ZrO2 and TiO2 in ionic form. The catalysts were synthesized by the solution combustion technique. The compounds showed high activity and CO tolerance for the reaction. The activity of Pd and Pt ion substituted TiO2 was comparable and was higher than Pd and Pt ion substituted ZrO2. The mechanisms of the reaction over the two supports were proposed by making use of the X-ray photoelectron spectroscopy and FT infrared spectroscopic observations. The reaction over ZrO2 supported catalysts was proposed to take place by the utilization of the surface hydroxyl groups while the reaction over TiO2 supported catalysts was hypothesized to be a hybrid mechanism utilizing surface hydroxyl groups and the lattice oxygen.

Insights into conformational and packing features in a series of aryl substituted ethyl-6-methyl-4-phenyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate

The supramolecular structures of eight aryl protected ethyl-6-methyl-4-phenyl-2-oxo-1,2,3,4-tetrahydropyrimidine- 5-carboxylates have been analyzed to determine the role of different functional groups on the molecular geometry, conformational characteristics and the packing of these molecules in the crystal lattice. Out of these the para fluoro substituted compound on the aryl ring exhibits conformational polymorphism, due to the different conformation of the ester moiety. This behaviour has been characterized using both powder and single-crystal X-ray diffraction, optical microscopy and differential scanning calorimetry performed on both these polymorphs. The compounds pack via the cooperative interplay of strong N-H center dot center dot center dot O=C intermolecular dimers and chains forming a sheet like structure. In addition, weak C-H center dot center dot center dot O=C and C-H center dot center dot center dot pi interactions impart additional stability to the crystal packing.

Metallo-beta-lactamase-Catalyzed Hydrolysis of Cephalosporins: Some Mechanistic Insights into the Effect of Heterocyclic Thiones on Enzyme Activity

The hydrolysis of beta-lactam antibiotics using zinc-containing metallo-beta-lactamases (m beta l) is one of the major bacterial defense systems. These enzymes can catalyze the hydrolysis of a variety of antibiotics including the latest generation of cephalosporins, cephamycins, and imipenem. It is shown in this paper that the cephalosporins having heterocyclic - SR side chains are less prone to m beta l-mediated hydrolysis than the antibiotics that do not have such side chains. This is partly due to the inhibition of enzyme activity by the thione moieties eliminated during hydrolysis. When the enzymatic hydrolysis of oxacillin was carried out in the presence of heterocyclic thiones such as MU, MDT, DMETT, and MMA, the catalytic activity of the enzyme was inhibited significantly by these compounds. Although the heterocyclic - SR moieties eliminated from the beta-lactams upon hydrolysis undergo a rapid tautomerism between thione and thiol forms, these compounds act as thiolate ligands toward zinc(II) ions. The structural characterization of two model tetranuclear zinc(II) thiolate complexes indicates that the -SR side chains eliminated from the antibiotics may interact with the zinc(II) metal center of m beta l through their sulfur atoms.

Crystal structure of Salmonella typhimurium 2-methylcitrate synthase: Insights on domain movement and substrate specificity

2-Methylcitric acid (2-MCA) cycle is one of the well studied pathways for the utilization of propionate as a source of carbon and energy in bacteria such as Salmonella typhimurium and Escherichia coli. 2-Methylcitrate synthase (2-MCS) catalyzes the conversion of oxaloacetate and propionyl-CoA to 2-methylcitrate and CoA in the second step of 2-MCA cycle. Here, we report the X-ray crystal structure of S. typhimurium 2-MCS (StPrpC) at 2.4 A resolution and its functional characterization. StPrpC was found to utilize propionyl-CoA more efficiently than acetyl-CoA or butyryl-CoA. The polypeptide fold and the catalytic residues of StPrpC are conserved in citrate synthases (CSs) suggesting similarities in their functional mechanisms. In the triclinic P1 cell, StPrpC molecules were organized as decamers composed of five identical dimer units. In solution, StPrpC was in a dimeric form at low concentrations and was converted to larger oligomers at higher concentrations. CSs are usually dimeric proteins. In Gram-negative bacteria, a hexameric form, believed to be important for regulation of activity by NADH, is also observed. Structural comparisons with hexameric E. coil CS suggested that the key residues involved in NADH binding are not conserved in StPrpC. Structural comparison with the ligand free and bound states of CSs showed that StPrpC is in a nearly closed conformation despite the absence of bound ligands. It was found that the Tyr197 and Leu324 of StPrpC are structurally equivalent to the ligand binding residues His and Val, respectively, of CSs. These substitutions might determine the specificities for acyl-CoAs of these enzymes. (C) 2010 Elsevier Inc. All rights reserved.

Halogen Bonding in 2,5-Dichloro-1,4-benzoquinone: Insights from Experimental and Theoretical Charge Density Analysis

Experimental charge density distribution in 2, 5-dichloro-1, 4-benzoquinone has been carried out using high resolution X-ray diffraction data at 90 K to quantitatively evaluate the nature of C-Cl center dot center dot center dot O=C halogen bond in molecular crystals. Additionally, the halogen bond is studied from geometrical point of view and the same has been visualized using Hirshfeld surface analysis. The obtained results from experimental charge density analysis are compared with periodic quantum calculations using B3LYP 6-31G(d,p) level of theory. The topological values at bond critical point, three-dimensional static deformation density features and electrostatic potential isosurfaces unequivocally establish the attractive nature of C-Cl center dot center dot center dot O=C halogen bond in crystalline lattice.

Insights into the Regulatory Characteristics of the Mycobacterial Dephosphocoenzyme A Kinase: Implications for the Universal CoA Biosynthesis Pathway

Being vastly different from the human counterpart, we suggest that the last enzyme of the Mycobacterium tuberculosis Coenzyme A biosynthetic pathway, dephosphocoenzyme A kinase (CoaE) could be a good anti-tubercular target. Here we describe detailed investigations into the regulatory features of the enzyme, affected via two mechanisms. Enzymatic activity is regulated by CTP which strongly binds the enzyme at a site overlapping that of the leading substrate, dephosphocoenzyme A (DCoA), thereby obscuring the binding site and limiting catalysis. The organism has evolved a second layer of regulation by employing a dynamic equilibrium between the trimeric and monomeric forms of CoaE as a means of regulating the effective concentration of active enzyme. We show that the monomer is the active form of the enzyme and the interplay between the regulator, CTP and the substrate, DCoA, affects enzymatic activity. Detailed kinetic data have been corroborated by size exclusion chromatography, dynamic light scattering, glutaraldehyde crosslinking, limited proteolysis and fluorescence investigations on the enzyme all of which corroborate the effects of the ligands on the enzyme oligomeric status and activity. Cysteine mutagenesis and the effects of reducing agents on mycobacterial CoaE oligomerization further validate that the latter is not cysteine-mediated or reduction-sensitive. These studies thus shed light on the novel regulatory features employed to regulate metabolite flow through the last step of a critical biosynthetic pathway by keeping the latter catalytically dormant till the need arises, the transition to the active form affected by a delicate crosstalk between an essential cellular metabolite (CTP) and the precursor to the pathway end-product (DCoA).

Thermodynamic analysis of enzyme catalysed reactions: new insights into the Michaelis-Menten equation

A simple thermodynamic analysis of the well-known Michaelis-Menten equation (MME) of enzyme catalysis is proposed that employs the chemical potential mu to follow the Gibbs free energy changes attending the formation of the enzyme-substrate complex and its turnover to the product. The main conclusion from the above analysis is that low values of the Michaelis constant KM and high values of the turnover number k(cat) are advantageous: this supports a simple algebraic analysis of the MME, although at variance with current thinking. Available data apparently support the above findings. It is argued that transition state stabilisation - rather than substrate distortion or proximity - is the key to enzyme catalysis.

Electrical switching in chalcogenide glasses - Some newer insights

The recent studies on the switching. behavior of several chalcogenide semiconductors indicate that there exists a close relation between the electrical switching and structural effects in these materials; the two network topological Thresholds, namely the Rigidity Percolation and the Chemical Threshold are found to influence considerably the composition dependence of the switching voltages/fields of many memory and threshold switching glasses. Further, changes in the coordination of constituent atoms are found to effect a change in the switching behavior (memory to threshold), Also, an interesting relation has been established between the type of switching exhibited and the thermal diffusivity of the material.

Molecular insights into the mechanism of phenotypic tolerance to rifampicin conferred on mycobacterial RNA polymerase by MsRbpA

The protein MsRbpA from Mycobacterium smegmatis rescues RNA polymerase (RNAP) from the inhibitory effect of rifampicin (Rif). We have reported previously that MsRbpA interacts with the beta-subunit of RNAP and that the effect of MsRbpA on Rif-resistant (Rif(R)) RNAP is minimal. Here we attempted to gain molecular insights into the mechanism of action of this protein with respect to its role in rescuing RNAP from Rif-mediated transcription inhibition. Our experimental approach comprised multiple-round transcription assays, fluorescence spectroscopy, MS and surface plasmon resonance in order to meet the above objective. Based on our molecular studies we propose here that Rif is released from its binding site in the RNAP-Rif complex in the presence of MsRbpA. Biophysical studies reveal that the location of MsRbpA on RNAP is at the junction of the beta- and beta'-subunits, close to the Rif-binding site and the (i + 1) site on RNAP.

The biological and structural characterization of Mycobacterium tuberculosis UvrA provides novel insights into its mechanism of action

Mycobacterium tuberculosis is an extremely well adapted intracellular human pathogen that is exposed to multiple DNA damaging chemical assaults originating from the host defence mechanisms. As a consequence, this bacterium is thought to possess highly efficient DNA repair machineries, the nucleotide excision repair (NER) system amongst these. Although NER is of central importance to DNA repair in M. tuberculosis, our understanding of the processes in this species is limited. The conserved UvrABC endonuclease represents the multi-enzymatic core in bacterial NER, where the UvrA ATPase provides the DNA lesion-sensing function. The herein reported genetic analysis demonstrates that M. tuberculosis UvrA is important for the repair of nitrosative and oxidative DNA damage. Moreover, our biochemical and structural characterization of recombinant M. tuberculosis UvrA contributes new insights into its mechanism of action. In particular, the structural investigation reveals an unprecedented conformation of the UvrB-binding domain that we propose to be of functional relevance. Taken together, our data suggest UvrA as a potential target for the development of novel anti-tubercular agents and provide a biochemical framework for the identification of small-molecule inhibitors interfering with the NER activity in M. tuberculosis.

Location and conformation of pantothenate and its derivatives in Mycobacterium tuberculosis pantothenate kinase: insights into enzyme action

Previous studies of complexes of Mycobacterium tuberculosis PanK (MtPanK) with nucleotide diphosphates and non-hydrolysable analogues of nucleoside triphosphates in the presence or the absence of pantothenate established that the enzyme has dual specificity for ATP and GTP, revealed the unusual movement of ligands during enzyme action and provided information on the effect of pantothenate on the location and conformation of the nucleotides at the beginning and the end of enzyme action. The X-ray analyses of the binary complexes of MtPanK with pantothenate, pantothenol and N-nonylpantothenamide reported here demonstrate that in the absence of nucleotide these ligands occupy, with a somewhat open conformation, a location similar to that occupied by phosphopantothenate in the `end' complexes, which differs distinctly from the location of pantothenate in the closed conformation in the ternary `initiation' complexes. The conformation and the location of the nucleotide were also different in the initiation and end complexes. An invariant arginine appears to play a critical role in the movement of ligands that takes place during enzyme action. The work presented here completes the description of the locations and conformations of nucleoside diphosphates and triphosphates and pantothenate in different binary and ternary complexes, and suggests a structural rationale for the movement of ligands during enzyme action. The present investigation also suggests that N-alkylpantothenamides could be phosphorylated by the enzyme in the same manner as pantothenate.