Insights into the Fold Organization of TIM Barrel from Interaction Energy Based Structure Networks

There are many well-known examples of proteins with low sequence similarity, adopting the same structural fold. This aspect of sequence-structure relationship has been extensively studied both experimentally and theoretically, however with limited success. Most of the studies consider remote homology or ``sequence conservation'' as the basis for their understanding. Recently ``interaction energy'' based network formalism (Protein Energy Networks (PENs)) was developed to understand the determinants of protein structures. In this paper we have used these PENs to investigate the common non-covalent interactions and their collective features which stabilize the TIM barrel fold. We have also developed a method of aligning PENs in order to understand the spatial conservation of interactions in the fold. We have identified key common interactions responsible for the conservation of the TIM fold, despite high sequence dissimilarity. For instance, the central beta barrel of the TIM fold is stabilized by long-range high energy electrostatic interactions and low-energy contiguous vdW interactions in certain families. The other interfaces like the helix-sheet or the helix-helix seem to be devoid of any high energy conserved interactions. Conserved interactions in the loop regions around the catalytic site of the TIM fold have also been identified, pointing out their significance in both structural and functional evolution. Based on these investigations, we have developed a novel network based phylogenetic analysis for remote homologues, which can perform better than sequence based phylogeny. Such an analysis is more meaningful from both structural and functional evolutionary perspective. We believe that the information obtained through the ``interaction conservation'' viewpoint and the subsequently developed method of structure network alignment, can shed new light in the fields of fold organization and de novo computational protein design.

Retrobiosynthetic Approach Delineates the Biosynthetic Pathway and the Structure of the Acyl Chain of Mycobacterial Glycopeptidolipids

Glycopeptidolipids (GPLs) are dominant cell surface molecules present in several non-tuberculous and opportunistic mycobacterial species. GPLs from Mycobacterium smegmatis are composed of a lipopeptide core unit consisting of a modified C-26-C-34 fatty acyl chain that is linked to a tetrapeptide (Phe-Thr-Ala-alaninol). The hydroxyl groups of threonine and terminal alaninol are further modified by glycosylations. Although chemical structures have been reported for 16 GPLs from diverse mycobacteria, there is still ambiguity in identifying the exact position of the hydroxyl group on the fatty acyl chain. Moreover, the enzymes involved in the biosynthesis of the fatty acyl component are unknown. In this study we show that a bimodular polyketide synthase in conjunction with a fatty acyl-AMP ligase dictates the synthesis of fatty acyl chain of GPL. Based on genetic, biochemical, and structural investigations, we determine that the hydroxyl group is present at the C-5 position of the fatty acyl component. Our retrobiosynthetic approach has provided a means to understand the biosynthesis of GPLs and also resolve the long-standing debate on the accurate structure of mycobacterial GPLs.

Spirodiazaselenuranes: synthesis, structure and antioxidant activity

In this paper, the synthesis, characterization and glutathione peroxidase and peroxynitrite scavenging activities of a series of stable spirodiazaselenuranes are described. The spiro compounds were synthesized in good yields by oxidative cyclization of diaryl selenides bearing amide moieties. All the selenides and spiro derivatives were characterized by H-1, C-13 and Se-77 NMR spectroscopy, mass spectral techniques and the structures of some of the spirodiazaselenuranes were confirmed by single crystal X-ray crystallography. The structures reveal that the selenium atom occupies the center of a distorted trigonal bipyramid core with two nitrogen atoms occupying the apical positions and two carbon atoms and the selenium lone pair occupying the equatorial positions. Mechanistic investigations indicate that the spirocyclization occurs via the formation of selenoxide intermediates. The new compounds were evaluated for their glutathione peroxidase (GPx) mimetic activity by using H2O2 as a substrate and glutathione (GSH) as a co-substrate. It was found that the substituents attached to the nitrogen atom of the selenazole ring have a significant effect on the GPx activity. While the introduction of electron withdrawing groups such as -Cl, -Br etc. to the phenyl ring decreases the activity, the introduction of electron donating groups such as -OH, -OMe significantly enhances the GPx activity of both diaryl selenides and spirodiazaselenuranes. In addition to GPx activity, the selenides and spiro derivatives were studied for their ability to inhibit peroxynitrite (PN)-mediated nitration of bovine serum albumin (BSA) and oxidation of dihydrorhodamine 123. These studies indicate that the diarylselenides effectively inhibit the PN-mediated nitration and oxidation reactions by reacting with PN to produce the corresponding spirodiazaselenuranes.

Structural and mechanistic investigations on Salmonella typhimurium acetate kinase (AckA): identification of a putative ligand binding pocket at the dimeric interface

Background: Bacteria such as Escherichia coli and Salmonella typhimurium can utilize acetate as the sole source of carbon and energy. Acetate kinase (AckA) and phosphotransacetylase (Pta), key enzymes of acetate utilization pathway, regulate flux of metabolites in glycolysis, gluconeogenesis, TCA cycle, glyoxylate bypass and fatty acid metabolism. Results: Here we report kinetic characterization of S. typhimurium AckA (StAckA) and structures of its unliganded (Form-I, 2.70 angstrom resolution) and citrate-bound (Form-II, 1.90 angstrom resolution) forms. The enzyme showed broad substrate specificity with k(cat)/K-m in the order of acetate > propionate > formate. Further, the K-m for acetyl-phosphate was significantly lower than for acetate and the enzyme could catalyze the reverse reaction (i.e. ATP synthesis) more efficiently. ATP and Mg2+ could be substituted by other nucleoside 5'-triphosphates (GTP, UTP and CTP) and divalent cations (Mn2+ and Co2+), respectively. Form-I StAckA represents the first structural report of an unliganded AckA. StAckA protomer consists of two domains with characteristic beta beta beta alpha beta alpha beta alpha topology of ASKHA superfamily of proteins. These domains adopt an intermediate conformation compared to that of open and closed forms of ligand-bound Methanosarcina thermophila AckA (MtAckA). Spectroscopic and structural analyses of StAckA further suggested occurrence of inter-domain motion upon ligand-binding. Unexpectedly, Form-II StAckA structure showed a drastic change in the conformation of residues 230-300 compared to that of Form-I. Further investigation revealed electron density corresponding to a citrate molecule in a pocket located at the dimeric interface of Form-II StAckA. Interestingly, a similar dimeric interface pocket lined with largely conserved residues could be identified in Form-I StAckA as well as in other enzymes homologous to AckA suggesting that ligand binding at this pocket may influence the function of these enzymes. Conclusions: The biochemical and structural characterization of StAckA reported here provides insights into the biochemical specificity, overall fold, thermal stability, molecular basis of ligand binding and inter-domain motion in AckA family of enzymes. Dramatic conformational differences observed between unliganded and citrate-bound forms of StAckA led to identification of a putative ligand-binding pocket at the dimeric interface of StAckA with implications for enzymatic function.

Investigations on structural and optical properties of co-doped (Ag, Co) ZnO nanoparticles

Undoped and co-doped (Ag, Co) ZnO powders were synthesized by chemical co-precipitation method without using any capping agent. The X-ray diffraction results indicate that the undoped and co-doped ZnO powders have pure hexagonal structure and are consisting of nanosized single-crystalline particles. The size of the nanoparticles increases with increasing Ag concentration from 1 to 5 mol% as compared to that of undoped ZnO. The presence of substitution dopants of Ag and Co in the ZnO host material was confirmed by the Energy dispersive analysis of X-rays (EDAX). Optical absorption measurements indicate blue shift and red-shift in the absorption band edge upon doping concentration of Ag and blue emission was observed by photoluminescence (PL) studies.

Discovery of novel glitazones incorporated with phenylalanine and tyrosine: Synthesis, antidiabetic activity and structure-activity relationships

We report a series of new glitazones incorporated with phenylalanine and tyrosine. All the compounds were tested for their in vitro glucose uptake activity using rat-hemidiaphragm, both in presence and absence of insulin. Six of the most active compounds from the in vitro screening were taken forward for their in vivo triglyceride and glucose lowering activity against dexamethazone induced hyperlipidemia and insulin resistance in Wistar rats. The liver samples of rats that received the most active compounds, 23 and 24, in the in vivo studies, were subjected to histopathological examination to assess their short term hepatotoxicity. The investigations on the in vitro glucose uptake, in vivo triglyceride and glucose lowering activity are described here along with the quantitative structure-activity relationships. (C) 2012 Elsevier Inc. All rights reserved.

Metabolites of PPI-2458, a selective, irreversible inhibitor of methionine aminopeptidase-2: structure determination and In vivo activity

The natural product fumagillin exhibits potent antiproliferative and antiangiogenic properties. The semisynthetic analog PPI-2458, (3R,4S,5S,6R)-5-methoxy-4-(2R,3R)-2-methyl-3-(3-methylbut-2-enyl) oxiran-2-yl]-1-oxaspiro2.5]octan-6-yl] N-(2R)-1-amino-3-methyl-1-oxobutan-2-yl]carbamate, demonstrates rapid inactivation of its molecular target, methionine aminopeptidase-2 (MetAP2), and good efficacy in several rodent models of cancer and inflammation with oral dosing despite low apparent oral bioavailability. To probe the basis of its in vivo efficacy, the metabolism of PPI-2458 was studied in detail. Reaction phenotyping identified CYP3A4/5 as the major source of metabolism in humans. Six metabolites were isolated from liver microsomes and characterized by mass spectrometry and nuclear resonance spectroscopy, and their structures were confirmed by chemical synthesis. The synthetic metabolites showed correlated inhibition of MetAP2 enzymatic activity and vascular endothelial cell growth. In an ex vivo experiment, MetAP2 inhibition in white blood cells, thymus, and lymph nodes in rats after single dosing with PPI-2458 and the isolated metabolites was found to correlate with the in vitro activity of the individual species. In a phase 1 clinical study, PPI-2458 was administered to patients with non-Hodgkin lymphoma. At 15 mg administered orally every other day, MetAP2 in whole blood was 80% inactivated for up to 48 hours, although the exposure of the parent compound was only similar to 10% that of the summed cytochrome P450 metabolites. Taken together, the data confirm the participation of active metabolites in the in vivo efficacy of PPI-2458. The structures define a metabolic pathway for PPI-2458 that is distinct from that of TNP-470 ((3R, 4S, 5S, 6R)-5-methoxy-4-(2R, 3R)-2-methyl-3-(3-methylbut-2-enyl)oxiran-2-yl]-1-oxaspiro2.5]octan-6 -yl] N-(2-chloroacetyl)carbamate). The high level of MetAP2 inhibition achieved in vivo supports the value of fumagillin-derived therapeutics for angiogenic diseases.

Microwave Spectroscopic and Atoms in Molecules Theoretical Investigations on the ArPropargyl Alcohol Complex: ArHO, Ar, and ArC Interactions

The structure of the Arpropargyl alcohol (ArPA) complex is determined from the rotational spectra of the parent complex and its two deuterated isotopologues, namely ArPA-D(OD) and ArPA-D(CD). The spectra confirm a geometry in which PA exists in the gauche form with Ar located in between OH and CCH groups. All a, b and c types of transitions show small splitting due to some large-amplitude motion dominated by COH torsion, as in the monomer. Splittings in a- and b-type transitions are of the order of a few kilohertz, whereas splitting in the c-type transitions is relatively larger (0.92.6 MHz) and decreases in the order ArPA>ArPA-D(CD)>ArPA-D(OD). The assignments are well supported by ab initio calculations. Atoms in molecules (AIM) and electrostatic potential calculations are used to explore the nature of the interactions in this complex. AIM calculations not only reveal the expected OHAr and Ar interactions in the Argauche-PA complex, but also novel CAr (of CH2OH group) and OHAr interactions in the Artrans-PA complex. Similar interactions are also present in the Armethanol complex.

Comprehensive investigations on DNa center dot center dot center dot A (D = H/F) complexes show why `sodium bonding' is not commonly observed

A comprehensive study of D-Na center dot center dot center dot A (D = H/F) complexes has been done using advanced ab initio and atoms in molecule (AIM) theoretical analyses. The correlation between electron density at bond critical point and binding energy gives a distinguishing feature for hydrogen bonding, different from the `electrostatic complexes' formed by LiD and NaD. Moreover, the LiD/NaD dimers have both linear and anti-parallel minima, as expected for electrostatic dipole-dipole interactions. The HF dimer has a quasi-linear minimum and the anti-parallel structure is a saddle point. Clearly, characterizing hydrogen bonding as `nothing but electrostatic interaction between two dipoles' is grossly in error.

Investigations on high enthalpy shock wave exposed graphitic carbon nanoparticles

The impact of high enthalpy shock wave on graphitic carbon nanoparticle (GCNP) films has been investigated and discussed in view of space and chemical engineering applications. The GCNP films were developed by using spray method and exposed to high enthalpy shock wave under an inert atmosphere. Upon shock wave treatment, two typical amendments such as weight loss in the deposited material and growth of second order nanostructures (SONS) have been observed. While increasing test gas pressure, the loss of material and density of SONs are gradually increased. Most of the shock wave induced SONS are highly crystalline and belong to the cubic diamond structure. Upon shock treatment as well as with increase of test gas pressure, a considerable improvement in the quality of GCNP films has been observed. Further, ablation of GCNPs exclusively on the top surface of the coatings and formation of hierarchical NPs (diamond NPs on GCNPs) has been observed.

Antithyroid drugs and their analogues: synthesis, structure, and mechanism of action

Thyroid hormones are essential for the development and differentiation of all cells of the human body. They regulate protein, fat, and carbohydrate metabolism. In this Account, we discuss the synthesis, structure, and mechanism of action of thyroid hormones and their analogues. The prohormone thyroxine (14) is synthesized on thyroglobulin by thyroid peroxidase (TPO), a heme enzyme that uses iodide and hydrogen peroxide to perform iodination and phenolic coupling reactions. The monodeiodination of T4 to 3,3',5-triiodothyronine (13) by selenium-containing deiodinases (ID-1, ID-2) is a key step in the activation of thyroid hormones. The type 3 deiodinase (ID-3) catalyzes the deactivation of thyroid hormone in a process that removes iodine selectively from the tyrosyl ring of T4 to produce 3,3',5'-triiodothyronine (rT3). Several physiological and pathological stimuli influence thyroid hormone synthesis. The overproduction of thyroid hormones leads to hyperthyroidism, which is treated by antithyroid drugs that either inhibit the thyroid hormone biosynthesis and/or decrease the conversion of T4 to T3. Antithyroid drugs are thiourea-based compounds, which indude propylthiouracil (PTU), methimazole (MM I), and carbimazole (CBZ). The thyroid gland actively concentrates these heterocyclic compounds against a concentration gradient Recently, the selenium analogues of PTU, MMI, and CBZ attracted significant attention because the selenium moiety in these compounds has a higher nucleophilicity than that of the sulfur moiety. Researchers have developed new methods for the synthesis of the selenium compounds. Several experimental and theoretical investigations revealed that the selone (C=Se) in the selenium analogues is more polarized than the thione (C=S) in the sulfur compounds, and the selones exist predominantly in their zwitterionic forms. Although the thionamide-based antithyroid drugs have been used for almost 70 years, the mechanism of their action is not completely understood. Most investigations have revealed that MMI and PTU irreversibly inhibit TPO. PTU, MTU, and their selenium analogues also inhibit ID-1, most likely by reacting with the selenenyl iodide intermediate. The good ID-1 inhibitory activity of Pill and its analogues can be ascribed to the presence of the -N(H)-C(=O)- functionality that can form hydrogen bonds with nearby amino add residues in the selenenyl sulfide state. In addition to the TPO and ID-1 inhibition, the selenium analogues are very good antioxidants. In the presence of cellular reducing agents such as GSH, these compounds catalytically reduce hydrogen peroxide. They can also efficiently scavenge peroxynitrite, a potent biological oxidant and nitrating agent.

Highly Stable Hexacoordinated Nonoxidovanadium(IV) Complexes of Sterically Constrained Ligands: Syntheses, Structure, and Study of Antiproliferative and Insulin Mimetic Activity

Three highly stable, hexacoordinated nonoxidovanadium(IV), V-IV(L)(2), complexes (1-3) have been isolated and structurally characterized with tridentate aroylhydrazonates containing ONO donor atoms. All the complexes are stable in the open air in the solid state as well as in solution, a phenomenon rarely observed in nonoxidovanadium(IV) complexes. The complexes have good solubility in organic solvents, permitting electrochemical and various spectroscopic investigations. The existence of nonoxidovanadium(IV) complexes was confirmed by elemental analysis, ESI mass spectroscopy, cyclic voltammetry, EPR, and magnetic susceptibility measurements. X-ray crystallography showed the N3O3 donor set to define a trigonal prismatic geometry in each case. All the complexes show in vitro insulin mimetic activity against insulin responsive L6 myoblast cells, with complex 3 being the most potent, which is comparable to insulin at the complex concentration of 4 mu M, while the others have moderate insulin mimetic activity. In addition, the in vitro antiproliferative activity of complexes 1-3 against the He La cell line was assayed. The cytotoxicity of the complexes is affected by the various functional groups attached to the bezoylhydrazone derivative and 2 showed considerable antiproliferative activity compared to the most commonly used chemotherapeutic drugs.

Investigations of Ramachandran disallowed conformations in protein domain families

In peptide and protein structures, occurrence of (phi,psi.) angles in the disallowed region of the Ramachandran map almost always suggests local regions of error or poor accuracy. However, very rarely genuine disallowed conformations occur as noted in the current study in proteins of known structure available at ultra-high resolution (<= 1.2 (A) over circle). In the current work, extent of conservation of genuine disallowed conformations in homologous proteins of known structures has been analyzed. From a dataset of 124 protein domain families, with structure of at least one constituent member in each family available at a resolution of 1.2 (A) over circle or better, we have analyzed the conservation of 221 disallowed conformations. It is observed that the disallowed conformation is only moderately conservedin protein domain families. In the gross dataset no particular residue type adopting disallowed conformation elicit high conservation of residue type though there are alignment positions in the dataset with complete conservation of both the residue type and the disallowed conformation. Conserved disallowed conformation in protein domain families play biologically significant role in roughly 50% of the cases. The residues with the disallowed conformation or its flanking residues are often located within or around the functional site of the protein. (C) 2013 Elsevier B.V. All rights reserved.

Multichannel-Emissive V-Shaped Boryl-BODIPY Dyads: Synthesis, Structure, and Remarkably Diverse Response toward Fluoride

Three new V-shaped boryl-BODIPY dyads (1-3) were synthesized and structurally characterized. Compounds 1-3 are structurally close molecular siblings differing only in the number of methyl substituents on the BODIPY moiety that were found to play a major role in determining their photophysical behavior. The dyads show rare forms of multiple-channel emission characteristics arising from different extents of electronic energy transfer (EET) processes between the two covalently linked fluorescent chromophores (borane and BODIPY units). Insights into the origin and nature of their emission behavior were gained from comparison with closely related model molecular systems and related photophysical investigations. Because of the presence of the Lewis acidic triarylborane moiety, the dyads function as highly selective and sensitive fluoride sensors with vastly different response behaviors. When fluoride binds to the tricoordinate borane center, dyad 1 shows gradual quenching of its BODIPY-dominated emission due to the ceasing of the (borane to BODIPY) EET process. Dyad 2 shows a ratiometric fluorescence response for fluoride ions. Dyad 3 forms fluoride-induced nanoaggregates that result in fast and effective quenching of its fluorescence intensity just for similar to 0.3 ppm of analyte (i.e., 0.1 equiv 0.26 ppm of fluoride). The small structural alterations in these three structurally close dyads (1 - 3) result in exceptionally versatile and unique photophysical behaviors and remarkably diverse responses toward a single analyte, i.e., fluoride ion.

FOLD TYPE II PYRIDOXAL 5 `-PHOSPHATE DEPENDENT ENZYMES: STRUCTURE, SUBSTRATE RECOGNITION AND CATALYSIS

Enzymes utilizing pyridoxal 5'-phosphate dependent mechanism for catalysis are observed in all cellular forms of living organisms. PLP-dependent enzymes catalyze a wide variety of reactions involving amino acid substrates and their analogs. Structurally, these ubiquitous enzymes have been classified into four major fold types. We have carried out investigations on the structure and function of fold type I enzymes serine hydroxymethyl transferase and acetylornithine amino transferase, fold type n enzymes catabolic threonine deaminase, D-serine deaminase, D-cysteine desulfhydrase and diaminopropionate ammonia lyase. This review summarizes the major findings of investigations on fold type II enzymes in the context of similar studies on other PLP-dependent enzymes. Fold type II enzymes participate in pathways of both degradation and synthesis of amino acids. Polypeptide folds of these enzymes, features of their active sites, nature of interactions between the cofactor and the polypeptide, oligomeric structure, catalytic activities with various ligands, origin of specificity and plausible regulation of activity are briefly described. Analysis of the available crystal structures of fold type II enzymes revealed five different classes. The dimeric interfaces found in these enzymes vary across the classes and probably have functional significance.