Neratur K. Lokanath

Structure of aldolase from Thermus thermophilus HB8 showing the contribution of oligomeric state to thermostability.

2-Deoxyribose-5-phosphate aldolase catalyzes a reversible aldol condensation of two aldehydes via formation of a covalent Schiff-base intermediate at the active lysine residue. The crystal structure of 2-deoxyribose-5-phosphate aldolase from Thermus thermophilus HB8 has been determined with and without the substrate at atomic resolution. This enzyme, which has a unique homotetramer structure, has been compared with the previously reported crystal structures of two orthologues from Escherichia coli and Aeropyrum pernix. In contrast to the similar alpha/beta-barrel fold of the monomers, substantial quaternary structural differences are observed between these three enzymes. Further comparison of the subunit-subunit interface areas of these aldolases showed a clear positive correlation between the interface area and the living temperature of the source organism. From these results, it is concluded that the oligomeric state of 2-deoxyribose-5-phosphate aldolase is important for the thermostability and not for the catalytic function.

Structural insights into the molecular mechanism of Escherichia coli SdiA, a quorum-sensing receptor

Escherichia coli SdiA is a quorum-sensing (QS) receptor that responds to autoinducers produced by other bacterial species to control cell division and virulence. Crystal structures reveal that E. coli SdiA, which is composed of an N-terminal ligand-binding domain and a C-terminal DNA-binding domain (DBD), forms a symmetrical dimer. Although each domain shows structural similarity to other QS receptors, SdiA differs from them in the relative orientation of the two domains, suggesting that its ligand-binding and DNA-binding functions are independent. Consistently, in DNA gel-shift assays the binding affinity of SdiA for the ftsQP2 promoter appeared to be insensitive to the presence of autoinducers. These results suggest that autoinducers increase the functionality of SdiA by enhancing the protein stability rather than by directly affecting the DNA-binding affinity. Structural analyses of the ligand-binding pocket showed that SdiA cannot accommodate ligands with long acyl chains, which was corroborated by isothermal titration calorimetry and thermal stability analyses. The formation of an intersubunit disulfide bond that might be relevant to modulation of the DNA-binding activity was predicted from the proximal position of two Cys residues in the DBDs of dimeric SdiA. It was confirmed that the binding affinity of SdiA for the uvrY promoter was reduced under oxidizing conditions, which suggested the possibility of regulation of SdiA by multiple independent signals such as quorum-sensing inducers and the oxidation state of the cell.

Structural basis for the reaction mechanism of UDP-glucose pyrophosphorylase

UDP-glucose pyrophosphorylases (UGPase; EC 2.7.7.9) catalyze the conversion of UTP and glucose-1-phosphate to UDP-glucose and pyrophosphate and vice versa. Prokaryotic UGPases are distinct from their eukaryotic counterparts and are considered appropriate targets for the development of novel antibacterial agents since their product, UDP-glucose, is indispensable for the biosynthesis of virulence factors such as lipopolysaccharides and capsular polysaccharides. In this study, the crystal structures of UGPase from Helicobacter pylori (HpUGPase) were determined in apo- and UDP-glucose/Mg2+-bound forms at 2.9 Å and 2.3 Å resolutions, respectively. HpUGPase is a homotetramer and its active site is located in a deep pocket of each subunit. Magnesium ion is coordinated by Asp130, two oxygen atoms of phosphoryl groups, and three water molecules with octahedral geometry. Isothermal titration calorimetry analyses demonstrated that Mg2+ ion plays a key role in the enzymatic activity of UGPase by enhancing the binding of UGPase to UTP or UDP-glucose, suggesting that this reaction is catalyzed by an ordered sequential Bi Bi mechanism. Furthermore, the crystal structure explains the specificity for uracil bases. The current structural study combined with functional analyses provides essential information for understanding the reaction mechanism of bacterial UGPases, as well as a platform for the development of novel antibacterial agents.

Recognition of oxovanadium(V) species and its separation from other metal species through selective complexation by some acyclic ligands

Acyclic molecules possessing -OH (phenoxo and alkoxo type) groups and imine or amine moieties have been developed to sense the specific preference for VO3+ species. These molecules also showed a capability to quantitatively separate oxovanadium(V) species from a reaction mixture containing metal species of V, Mo, U, Fe, and Mn ions in solution. A cascade quantitative separation of VO3+ followed by cis-MoO2+2 followed by trans UO2+2 species is demonstrated from their mixture. Synthesis and structural details of oxo-species of vanadium molybdenum and uranium are also discussed. Factors influencing the complexation of these molecules towards oxo metal species of V, Mo and U are also addressed. 1999 Elsevier Science Ltd. All rights reserved.

Design, synthesis, anticonvulsant and analgesic studies of new pyrazole analogues: a Knoevenagel reaction approach

The present work involves the design and synthesis of a number of new compounds starting from 3-(3,4-dihalophenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde. The compounds were synthesized by adopting the Knoevenagel condensation reaction to meet the structural prerequisite required for anticonvulsant and analgesic activities. The reaction of 3-(3,4-dihalophenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde with substituted thiazolidine, pyrazolone, thiazolo[3,2-a]pyrimidine, Meldrums acid and barbituric acid yielded a variety of heterocycles bearing the pyrazole moiety. The newly synthesized compounds were characterized by elemental and spectroscopic analysis; in addition, the structure of compound 1a has been elucidated by single crystal X-ray diffraction technique. The synthesized molecules were evaluated for their in vivo anticonvulsant activity using maximal electroshock seizure (MES) test, while their analgesic activity was investigated by tail flick method. Further, rotarod toxicity method was used to study the toxicity profile of the selected compounds. Among the synthesized compounds, 1a, 4a and 7a possessed potent anticonvulsant activity and 1b, 5a, 5b, 7a and 7b showed the highest analgesic activity without displaying any toxicity. Efforts were also made to establish structure–activity relationships among the tested compounds.

Crystal structure of a component of glycine cleavage system: T-protein from Pyrococcus horikoshii OT3 at 1.5 Å resolution

Introduction. The glycine cleavage system (GCS; EC 2.1.2.10) is a multicomponent enzyme system that catalyzes the cleavage of glycine to yield carbon dioxide, ammonia, the methylene carbon unit in 5,10-CH2-tetrahydrofolate, and reduced nicotinamide adenine dinucleotide (NADH) H . This system is composed of 4 proteins, namely, a pyridoxal phosphate enzyme (P-protein), a carrier protein containing covalently bound lipoic acid (H-protein), a folate dependent enzyme (T-protein), and a protein exhibiting lipoamide dehydrogenase activity (Lprotein). The oxidative cleavage of glycine, catalyzed by the GCS system, is a major catabolic pathway in various organisms. In the first step, the P-protein catalyzes the decarboxylation of the glycine molecule and subsequent transfer of the residual methylamine to the oxidized H-protein (Hox), generating the methylamine-loaded Hprotein (Hmet). In the second step, the T-protein, a folate dependent enzyme, catalyzes the transfer of a methylene carbon from Hmet to tetrahydrofolate, resulting in the release of ammonia and the generation of reduced Hprotein (Hred). In the last step, the hydrolipoyl group of Hred is oxidized by the L-protein and Hox is generated, thereby completing the catalytic cycle. These sequential reactions are reversible. The biochemical properties of GCS in bacteria and mitochondria of plants and mammals have been extensively studied. In humans, a genetic disease termed nonketotic hyperglycinemia, caused by the absence of CGS (due to a number of mutations in the T-protein), results in a dramatic accumulation of glycine in blood, leading to metabolic and neurological disorders. Although the H-protein and the L-protein from several sources have been well studied by X-ray crystallography, the three-dimensional (3D) structures of the Pprotein and the T-protein have not yet been reported. Here, we report the crystal structure of the T-protein from Pyrococcus horikoshii OT3.

Design and environmentally benign synthesis of novel thiophene appended pyrazole analogues as anti-inflammatory and radical scavenging agents: Crystallographic, in silico modeling, docking and SAR characterization

Oxidative-stress induces inflammatory diseases and infections caused by drug-resistant microbial strains are on the rise necessitating the discovery of novel small-molecules for intervention therapy. The current study presents an effective and new green protocol for the synthesis of thiophene-appended pyrazoles through 3+2 annulations method. Chalcones 3(a-g) were prepared from 5-chloro-2-acetylthiophene and aromatic aldehydes by Claisen-Schmidt approach. The reaction of chalcones 3(a-g) with phenylhydrazine hydrochlorides 4(a-b) in acetic acid (30%) medium and also with freshly prepared citrus extract medium under reflux conditions produced the thiophene appended pyrazoles 5(a-l) in moderate yields. Structures of synthesized new pyrazoles were confirmed by spectral studies, elemental analysis and single crystal X-ray diffraction studies. Further, preliminary assessment of the anti-inflammatory properties of the compounds showed that, amongst the series, compounds 5d, 5e and 5l have excellent anti-inflammatory activities. Further, compounds 5c, 5d, 5g, and 5i exhibited excellent DPPH radical scavenging abilities in comparison with the standard ascorbic acid. Furthermore, using detailed structural modeling and docking efforts, combined with preliminary SAR, we show possible structural and chemical features on both the small-molecules and the protein that might contribute to the binding and inhibition.

Purification, crystallization and preliminary crystallographic analysis of the glycine-cleavage system component T-protein from Pyrococcus horikoshii OT3

The glycine-cleavage system component T-protein is a folate-dependent enzyme that catalyzes the formation of ammonia and 5,10-CH2-tetrahydrofolate from the aminomethyl intermediate bound to the lipoate cofactor of H-protein. T-protein from Pyrococcus horikoshii OT3 has been cloned, overexpressed in Escherichia coli, purified and crystallized by the microbatch method using PEG 4000 as a precipitant at 296 K. X-ray diffraction data have been collected to 1.50 A resolution at 100 K using synchrotron radiation. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 78.980, b = 95.708, c = 118.331 A. Assuming one homodimer per asymmetric unit gives a VM value of 2.4 A3 Da(-1) and a solvent content of 49.0%.

Addition of NOCl to cyclic vinylsilanes: An unexpected reversal of regiochemistry

NOCl adds to cyclic vinylsilanes in a syn manner with NO+ bonding to the beta -carbon and Cl- to the alpha -carbon, which is a reversal of the regiochemistry expected from the beta -silicon effect. The adducts dimerize to a single diastereomer containing enantiomeric pairs and/or give secondary products on further reaction

Crystal Structure of an Unsymmetrical Dimeric Liquid Crystal with a Wide Temperature Range Chiral Smectic A Phase

Abstract Cholesteryl 6-{4-[4-(1S-methylheptyloxy)phenylethynyl]phenoxy} hexanoate, C110H160O8, crystallises in the monoclinic space group P21 with a = 18.710(12)Å, b = 9.740(14)Å, c = 27.865(16)Å, α = 90.00˚, β = 103.85(5)˚, γ = 90.00˚, V = 4931(8)Å3 Z = 2, D(cal) = 1.085Mg/m3, μ = 0.066 mm−1, F 000 = 1768, GooF = 0.83, R1 = 0.07 and wR2 = 0.20.