The Evolution of Guanylyl Cyclases as Multidomain Proteins: Conserved Features of Kinase-Cyclase Domain Fusions

Guanylyl cyclases (GCs) are enzymes that generate cyclic GMP and regulate different physiologic and developmental processes in a number of organisms. GCs possess sequence similarity to class III adenylyl cyclases (ACs) and are present as either membrane-bound receptor GCs or cytosolic soluble GCs. We sought to determine the evolution of GCs using a large-scale bioinformatic analysis and found multiple lineage-specific expansions of GC genes in the genomes of many eukaryotes. Moreover, a few GC-like proteins were identified in prokaryotes, which come fused to a number of different domains, suggesting allosteric regulation of nucleotide cyclase activity Eukaryotic receptor GCs are associated with a kinase homology domain (KHD), and phylogenetic analysis of these proteins suggest coevolution of the KHD and the associated cyclase domain as well as a conservation of the sequence and the size of the linker region between the KHD and the associated cyclase domain. Finally, we also report the existence of mimiviral proteins that contain putative active kinase domains associated with a cyclase domain, which could suggest early evolution of the fusion of these two important domains involved in signa transduction.

Allostery in tRNA Synthetases Elucidated from MD Simulations and Protein Structure Networks

tRNA synthetases (aaRS) are enzymes crucial in the translation of genetic code. The enzyme accylates the acceptor stem of tRNA by the congnate amino acid bound at the active site, when the anti-codon is recognized by the anti-codon site of aaRS. In a typical aaRS, the distance between the anti-codon region and the amino accylation site is approximately 70 Å. We have investigated this allosteric phenomenon at molecular level by MD simulations followed by the analysis of protein structure networks (PSN) of non-covalent interactions. Specifically, we have generated conformational ensembles by performing MD simulations on different liganded states of methionyl tRNA synthetase (MetRS) from Escherichia coli and tryptophenyl tRNA synthetase (TrpRS) from Human. The correlated residues during the MD simulations are identified by cross correlation maps. We have identified the amino acids connecting the correlated residues by the shortest path between the two selected members of the PSN. The frequencies of paths have been evaluated from the MD snapshots[1]. The conformational populations in different liganded states of the protein have been beautifully captured in terms of network parameters such as hubs, cliques and communities[2]. These parameters have been associated with the rigidity and plasticity of the protein conformations and can be associated with free energy landscape. A comparison of allosteric communication in MetRS and TrpRS [3] elucidated in this study highlights diverse means adopted by different enzymes to perform a similar function. The computational method described for these two enzymes can be applied to the investigation of allostery in other systems.

Beta-Ketoacyl-ACP Synthase I/II from Plasmodium falciparum (PfFabB/F)-Is it B or F?

Condensing enzymes play an important and decisive role in terms of fatty acid composition of any organism. They can be classified as condensing enzymes involved in initiating the cycle and enzymes involved in elongating the initiated fatty acyl chain. In E. coli, two isoforms for the elongation condensing enzymes (FabB and FabF) exists whereas Plasmodium genome contains only one isoform. By in vitro complementation studies in E. coli CY244 cells, we show that PfFabB/ functions like E. coli FabF as the growth of the mutant cells could rescued only in the presence of oleic acid. But unlike bacterial enzyme, PfFabB/F does not increase the cis-vaccenic acid content in the mutant cells upon lowering the growth temperature. This study thus highlights the distinct properties of P. falciparum FabF which sets it apart from E. coli and most other enzymes of this family, described so far.

Generation of H2O2 in biomembranes

Knowledge of the generation of H202 in cellular oxidations has existed for many years. It has been assumed that H202 is tOxiC tO cells and the presence of catalase is indicative of a detoxication mechanism. Other radicals of oxygen were recently recognized to be more potent destructive agents of biological material than H202. Also catalase and other peroxidases utilize H202 in some cellular oxidation processes leading to several important metabolites. Thus, the generation of H202 in cellular processes seems to be purposeful and H202 can not be dismissed as a mere undesirable byproduct. Biological formation of H202 is not limited to the previously known flavoproteins and some copper enzymes, but other redox systems, particularly heme and non-heme iron proteins, are now found to undergo auto-oxidation yielding H202. The capacity for generation of H202 is now found to be widespread in a variety of organisms and in the organdies of the cells. The reduction of oxygen to H20 by mitochondrial cytochrome oxidase being the predominant oxygen-utilizing reaction had over-shadowed the importance of the quantitatively minor pathways. Under aerobic conditions generation of H202 by a Variety of biomembranes has now been found to be a physiological event interlinked with phenomena such as phagocytosis, transport processes and thermogenesis in some as yet unidentified way. The underlying mechanisms of these processes seem to involve generation and utilization of H202 in mitochondria, microsomes, peroxisomes or plasma membranes. This review gives an account of the potential of biomembranes to generate H202 and its implication in the cellular processes.

Interaction of Cibacron Blue F3G-A and Procion Red HE-3B with sheep liver 5,10-methylenetetrahydrofolate reductase

Cibacron Blue F3G-A, a probe used to monitor nucleotide binding domains in enzymes, inhibited sheep liver 5, 10-methylenetetrahydrofolate reductase competitively with respect to 5-methyltetrahydrofolate and NADPH. The Ki values obtained by kinetic methods and the Kd value for the binding of the dye to the enzyme estimated by protein fluorescence quenching were in the range 0·9-1·2 μM. Another triazine dye, Procion Red HE-3B interacted with the enzyme in an essentially similar manner to that observed with Cibacron Blue F3G-A. These results as well as the interaction of the dye with the enzyme monitored by difference spectroscopy and intrinsic protein fluorescence quenching methods indicated that the dye was probably interacting at the active site of the enzyme by binding at a hydrophobic region.

Noble Metal Ionic Catalysts

Because of growing environmental concerns and increasingly stringent regulations governing auto emissions, new more efficient exhaust catalysts are needed to reduce the amount of pollutants released from internal combustion engines. To accomplish this goal, the major pollutants in exhaust-CO, NOx, and unburned hydrocarbons-need to be fully converted to CO2, N-2, and H2O. Most exhaust catalysts contain nanocrystalline noble metals (Pt, Pd, Rh) dispersed on oxide supports such as Al2O3 or SiO2 promoted by CeO2. However, in conventional catalysts, only the surface atoms of the noble metal particles serve as adsorption sites, and even in 4-6 nm metal particles, only 1/4 to 1/5 of the total noble metal atoms are utilized for catalytic conversion. The complete dispersion of noble metals can be achieved only as ions within an oxide support. In this Account, we describe a novel solution to this dispersion problem: a new solution combustion method for synthesizing dispersed noble metal ionic catalysts. We have synthesized nanocrystalline, single-phase Ce1-xMxO2-delta and Ce1-x-yTiyMxO2-delta (M = Pt, Pd, Rh; x = 0,01-0.02, delta approximate to x, y = 0.15-0.25) oxides in fluorite structure, In these oxide catalysts, pt(2+), Pd2+, or Rh3+ ions are substituted only to the extent of 1-2% of Ce4+ ion. Lower-valent noble metal ion substitution in CeO2 creates oxygen vacancies. Reducing molecules (CO, H-2, NH3) are adsorbed onto electron-deficient noble metal ions, while oxidizing (02, NO) molecules are absorbed onto electron-rich oxide ion vacancy sites. The rates of CO and hydrocarbon oxidation and NOx reduction (with >80% N-2 selectivity) are 15-30 times higher in the presence of these ionic catalysts than when the same amount of noble metal loaded on an oxide support is used. Catalysts with palladium ion dispersed in CeO2 or Ce1-xTixO2 were far superior to Pt or Rh ionic catalysts. Therefore, we have demonstrated that the more expensive Pt and Rh metals are not necessary in exhaust catalysts. We have also grown these nanocrystalline ionic catalysts on ceramic cordierite and have reproduced the results we observed in powder material on the honeycomb catalytic converter. Oxygen in a CeO2 lattice is activated by the substitution of Ti ion, as well as noble metal ions. Because this substitution creates longer Ti-O and M-O bonds relative to the average Ce-O bond within the lattice, the materials facilitate high oxygen storage and release. The interaction among M-0/Mn+, Ce4+/Ce3+, and Ti4+/Ti3+ redox couples leads to the promoting action of CeO2, activation of lattice oxygen and high oxygen storage capacity, metal support interaction, and high rates of catalytic activity in exhaust catalysis.

Styrene Oxidation to Benzaldehyde by Polymer Anchored Wilkinson Complex

Wilkinson complex, insolubilized by anchoring to polymeric Amberlite beads, had been used for the liquid-phase catalytic oxidation of styrene to benzaldehyde and formaldehyde in toluene medium. Styrene conversion was followed by measuring the oxygen volume in contact with the reaction mixture in a specially designed closed batch apparatus. Styrene conversion depended upon catalyst loading and distribution inside the porous beads, while temperature had little effect on it. The internal diffusional effects on the conversion process have been taken into consideration by a mathematical model which allowed calculation of effectiveness factors for various catalyst loadings and corresponding catalyst distributions. The influence of external diffusion was separately determined by plotting initial rate versus catalyst loading. The proposed method can be readily extended to immobilized enzymes in porous matrices.

Styrene Oxidation to Benzaldehyde by Polymer Anchored Wilkinson Complex

Wilkinson complex, insolubilized by anchoring to polymeric Amberlite beads, had been used for the liquid-phase catalytic oxidation of styrene to benzaldehyde and formaldehyde in toluene medium. Styrene conversion was followed by measuring the oxygen volume in contact with the reaction mixture in a specially designed closed batch apparatus. Styrene conversion depended upon catalyst loading and distribution inside the porous beads, while temperature had little effect on it. The internal diffusional effects on the conversion process have been taken into onsideration by a mathematical model which allowed calculation of effectiveness factors for various catalyst loadings and corresponding catalyst distributions. The influence of external diffusion was separately determined by plotting initial rate versus catalyst loading. The proposed method can be readily extended to immobilized enzymes in porous matrices.

Purification and physicochemical, kinetic and immunological properties of allosteric serine hydroxymethyltransferase from monkey liver.

The homogeneous serine hydroxymethyltransferase purified from monkey liver, by the use of Blue Sepharose affinity chromatography, exhibited positive homotropic co-operative interactions (h = 2.5) with tetrahydrofolate and heterotropic interactions with L-serine and nicotinamide nucleotides. The enzyme had an unusually high temperature optimum of 60 degrees C and was protected against thermal inactivation by L-serine. The allosteric effects were abolished when the monkey liver enzyme was purified by using a heat-denaturation step in the presence of L-serine, a procedure adopted by earlier workers for the purification of this enzyme from mammalian and bacterial sources. The enzyme activity was inhibited completely by N5-methyltetrahydrofolate, N5-formyltetrahydrofolate, dichloromethotrexate, aminopterin and D-cycloserine, whereas methotrexate and dihydrofolate were partial inhibitors. The insoluble monkey liver enzyme-antibody complex was catalytically active and failed to show positive homotropic co-operative interactions with tetrahydrofolate (h = 1) and heterotropic interactions with NAD+. The enzyme showed a higher heat-stability in a complex with its antibody than as the free enzyme. These results highlight the pitfalls in using a heat-denaturation step in the purification of allosteric enzymes.

Structural effects of a dimer interface mutation on catalytic activity of triosephosphate isomerase.The role of conserved residues and complementary mutations

The active site of triosephosphate isomerase (TIM, EC: 5.3.1.1), a dimeric enzyme, lies very close to the subunit interface. Attempts to engineer monomeric enzymes have yielded well-folded proteins with dramatically reduced activity. The role of dimer interface residues in the stability and activity of the Plasmodium falciparum enzyme, PfTIM, has been probed by analysis of mutational effects at residue 74. The PfTIM triple mutant W11F/W168F/Y74W (Y74W*) has been shown to dissociate at low protein concentrations, and exhibits considerably reduced stability in the presence of denaturants, urea and guanidinium chloride. The Y74W* mutant exhibits concentration-dependent activity, with an approximately 22-fold enhancement of kcat over a concentration range of 2.5–40 μm, suggesting that dimerization is obligatory for enzyme activity. The Y74W* mutant shows an approximately 20-fold reduction in activity compared to the control enzyme (PfTIM WT*, W11F/W168F). Careful inspection of the available crystal structures of the enzyme, together with 412 unique protein sequences, revealed the importance of conserved residues in the vicinity of the active site that serve to position the functional K12 residue. The network of key interactions spans the interacting subunits. The Y74W* mutation can perturb orientations of the active site residues, due to steric clashes with proximal aromatic residues in PfTIM. The available crystal structures of the enzyme from Giardia lamblia, which contains a Trp residue at the structurally equivalent position, establishes the need for complementary mutations and maintenance of weak interactions in order to accommodate the bulky side chain and preserve active site integrity.

Analysis of proteins with the `hot dog' fold: Prediction of function and identification of catalytic residues of hypothetical proteins

Background: The hot dog fold has been found in more than sixty proteins since the first report of its existence about a decade ago. The fold appears to have a strong association with fatty acid biosynthesis, its regulation and metabolism, as the proteins with this fold are predominantly coenzyme A-binding enzymes with a variety of substrates located at their active sites. Results: We have analyzed the structural features and sequences of proteins having the hot dog fold. This study reveals that though the basic architecture of the fold is well conserved in these proteins, significant differences exist in their sequence, nature of substrate and oligomerization. Segments with certain conserved sequence motifs seem to play crucial structural and functional roles in various classes of these proteins. Conclusion: The analysis led to predictions regarding the functional classification and identification of possible catalytic residues of a number of hot dog fold-containing hypothetical proteins whose structures were determined in high throughput structural genomics projects.

Effect of L-cystine on macromolecular changes during spore and parasporal crystal formation inBacillus thuringiensis var.thuringiensis

High concentration of L-cystine (0.25%) when present in a glucose-mineral salt medium inhibited sporulation-specific events like protease production, calcium uptake and dipicolinic acid synthesis inBacillus thuringiensis var.thuringiensis. In addition, the enzymes of the Krebs cycle from aconitase onwards were completely inhibited by a high concentration of cystine. At a low concentration of cystine (0.05%), none of the above mentioned macromolecular changes were affected. Lipid synthesis monitored by [1,214 C]-acetate incorporation into lipid as well as into whole cells was completely inhibited.

Metallomicelles as potent catalysts for the ester hydrolysis reactions in water

Synthetic amphiphiles have been employed for the investigation of diverse topics, e.g. membrane mimetics, drug delivery, ion sensing and even in certain separation processes. Metal-complexing amphiphiles comprise an interesting class of compounds possessing multiple utilities. Upon solubilization in water they form metallomicelles. For achieving specific catalysis of a variety of reactions, metallomicelles were utilized by applying the principles of coordination chemistry and self-organizing systems. Because of their certain similarities with the natural enzymes, metallomicelles were synthesized as catalysts for many reactions. In particular the metallomicelles play a catalytic role in reactions involving the hydrolysis of activated carboxylate esters, phosphate esters and amides at ambient conditions near neutral pH. Apart from the hydrolysis reactions, these were exploited to play pertinent role as Lewis acid catalysts in cycloaddition reactions, and in other reactions such as phenolic oxidation in presence of hydrogen peroxide. In this review we emphasize with the help of assorted examples, the design, synthesis of metal-complexing amphiphiles and their aggregation behavior leading to catalytic hydrolysis reactions in aqueous media.

Inhibition of monkey liver serine hydroxymethyltransferase by Cibacron Blue 3G-A

Cibacron Blue 3G-A inhibited monkey liver serine hydroxymethyltransferase competitively with respect to tetrahydrofolate and non-competitively with respect to L-serine. NADH, a positive heterotropic effector, failed to protect the enzymes against inhibition by the dye and was unable to desorb the enzyme from Blue Sepharose CL-6B gel matrix. The binding of the dye to the free enzyme was confirmed by changes in the dye absorption spectrum. The results indicate that the dye probably binds at the tetrahydrofolate-binding domain of the enzyme, rather than at the 'dinucleotide fold'.

Role of conformational dynamics in kinetics of an enzymatic cycle in a nonequilibrium steady state

Enzyme is a dynamic entity with diverse time scales, ranging from picoseconds to seconds or even longer. Here we develop a rate theory for enzyme catalysis that includes conformational dynamics as cycling on a two-dimensional (2D) reaction free energy surface involving an intrinsic reaction coordinate (X) and an enzyme conformational coordinate (Q). The validity of Michaelis-Menten (MM) equation, i.e., substrate concentration dependence of enzymatic velocity, is examined under a nonequilibrium steady state. Under certain conditions, the classic MM equation holds but with generalized microscopic interpretations of kinetic parameters. However, under other conditions, our rate theory predicts either positive (sigmoidal-like) or negative (biphasic-like) kinetic cooperativity due to the modified effective 2D reaction pathway on X-Q surface, which can explain non-MM dependence previously observed on many monomeric enzymes that involve slow or hysteretic conformational transitions. Furthermore, we find that a slow conformational relaxation during product release could retain the enzyme in a favorable configuration, such that enzymatic turnover is dynamically accelerated at high substrate concentrations. The effect of such conformation retainment in a nonequilibrium steady state is evaluated.