Preliminary X-ray crystallographic studies on acetate kinase (AckA) from Salmonella typhimurium in two crystal forms

Acetate kinase (AckA) catalyzes the reversible transfer of a phosphate group from acetyl phosphate to ADP, generating acetate and ATP, and plays a central role in carbon metabolism. In the present work, the gene corresponding to AckA from Salmonella typhimurium (StAckA) was cloned in the IPTG-inducible pRSET C vector, resulting in the attachment of a hexahistidine tag to the N-terminus of the expressed enzyme. The recombinant protein was overexpressed, purified and crystallized in two different crystal forms using the microbatch-under-oil method. Form I crystals diffracted to 2.70 angstrom resolution when examined using X-rays from a rotating-anode X-ray generator and belonged to the monoclinic space group C2, with unit-cell parameters a = 283.16, b = 62.17, c = 91.69 angstrom, beta = 93.57 degrees. Form II crystals, which diffracted to a higher resolution of 2.35 angstrom on the rotating-anode X-ray generator and to 1.90 angstrom on beamline BM14 of the ESRF, Grenoble, also belonged to space group C2 but with smaller unit-cell parameters (a = 151.01, b = 78.50, c = 97.48 angstrom, beta = 116.37 degrees). Calculation of Matthews coefficients for the two crystal forms suggested the presence of four and two protomers of StAckA in the asymmetric units of forms I and II, respectively. Initial phases for the form I diffraction data were obtained by molecular replacement using the coordinates of Thermotoga maritima AckA (TmAckA) as the search model. The form II structure was phased using a monomer of form I as the phasing model. Inspection of the initial electron-density maps suggests dramatic conformational differences between residues 230 and 300 of the two crystal forms and warrants further investigation.

Crystal structure of a beta-prism II lectin from Remusatia vivipara

The crystal structure of a beta-prism II (BP2) fold lectin from Remusatia vivipara, a plant of traditional medicinal value, has been determined at a resolution of 2.4 A. This lectin (RVL, Remusatia vivipara lectin) is a dimer with each protomer having two distinct BP2 domains without a linker between them. It belongs to the ``monocot mannose-binding'' lectin family, which consists of proteins of high sequence and structural similarity. Though the overall tertiary structure is similar to that of lectins from snowdrop bulbs and garlic, crucial differences in the mannose-binding regions and oligomerization were observed. Unlike most of the other structurally known proteins in this family, only one of the three carbohydrate recognition sites (CRSs) per BP2 domain is found to be conserved. RVL does not recognize simple mannose moieties. RVL binds to only N-linked complex glycans like those present on the gp120 envelope glycoprotein of HIV and mannosylated blood proteins like fetuin, but not to simple mannose moieties. The molecular basis for these features and their possible functional implications to understand the different levels of carbohydrate affinities in this structural family have been investigated through structure analysis, modeling and binding studies. Apart from being the first structure of a lectin to be reported from the Araceae/Arum family, this protein also displays a novel mode of oligomerization among BP2 lectins.

Benzothiophene Carboxamide Derivatives as Inhibitors of Plasmodium falciparum Enoyl-ACP Reductase

Benzothiophene derivatives like benzothiophene sulphonamides, biphenyls, or carboxyls have been synthesized and have found wide pharmacological usage. Here we report, bromo-benzothiophene carboxamide derivatives as potent, slow tight binding inhibitors of Plasmodium enoyl-acyl carrier protein (ACP) reductase (PfENR). 3-Bromo-N-(4-fluorobenzyl)-benzo[b]thiophene-2-carboxamide (compound 6) is the most potent inhibitor with an IC(50) of 115 nM for purified PfENR. The inhibition constant (K(i)) of compound 6 was 18 nM with respect to the cofactor and 91 nM with respect to crotonoyl-CoA. These inhibitors showed competitive kinetics with cofactor and uncompetitive kinetics with the substrate. Thus, these compounds hold promise for the development of potent antimalarials. (C) 2011 IUBMB IUBMB Life, 63(12): 1101-1110, 2011

Benzothiophene Carboxamide Derivatives as Novel Antimalarials

Bromo-benzothiophene carboxamide derivatives have been shown in the preceding article to inhibit Plasmodium falciparum Enoyl-ACP reductase. Here, we report bromo-benzothiophene carboxamide derivatives as potent inhibitors of Plasmodium asexual blood-stages in vitro as well as in vivo in the mouse model. These compounds specifically impair the development of metabolically active trophozoite stage of intraerythrocytic cycle and the intravenous administration of 3-bromo-N-(4-fluorobenzyl)-benzo[b]thiophene-2-carboxamide (compound 6) enhances the longevity of P. berghei infected mice by 2 weeks compared to disease control animals thereby preventing the onset of ataxia and convulsions in treated mice. These compounds thus hold promise for the development of potent antimalarials.(C) 2011 IUBMB IUBMB Life, 63(12): 1111-1115, 2011

l-pGlu-(2-propyl)-l-His-l-ProNH2 attenuates 4-aminopyridine-induced epileptiform activity and sodium current: a possible action of new thyrotropin-releasing hormone analog for its anticonvulsant potential

L-PGlu-(2-proPyl)-L-His-L-ProNH(2) (NP-647) is a CNS active thyrotropin-releasing hormone (TRH) analog with potential application in various CNS disorders including seizures. In the present study, mechanism of action for protective effect of NP-647 was explored by studying role of NP-647 on epileptiform activity and sodium channels by using patch-clamp methods. Epileptiform activity was induced in subicular pyramidal neurons of hippocampal slice of rat by perfusing 4-aminopyridine (4-AP) containing Mg(+2)-free normal artificial cerebrospinal fluid (nACSF). Increase in mean firing frequency was observed after perfusion of 4-AP and zero Mg(+2) (2.10+/-0.47 Hz) as compared with nACSF (0.12+/-0.08 Hz). A significant decrease in mean firing frequency (0.61+/-0.22 Hz), mean frequency of epileptiform events (0.03+/-0.02 Hz vs. 0.22+/-0.05 Hz of 4-AP+0 Mg), and average number of action potentials in paroxysmal depolarization shift-burst (2.54+/-1.21 Hz vs. 8.16+/-0.88 Hz of 4-AP +0 Mg) was observed. A significant reduction in peak dV/dt (246+/-19 mV ms(-1) vs. 297 18 mV ms-1 of 4-AP+0 Mg) and increase (1.332+/-0.018 ms vs. 1.292+/-0.019 ms of 4-AP+0 Mg) in time required to reach maximum depolarization were observed indicating role of sodium channels. Concentration-dependent depression of sodium current was observed after exposure to dorsal root ganglion neurons to NP-647. NP-647 at different concentrations (1, 3, and 10 mu M) depressed sodium current (15+/-0.5%, 50+/-2.6%, and 75+/-0.7%, respectively). However, NP-647 did not show change in the peak sodium current in CNa18 cells. Results of present study demonstrated potential of NP-647 in the inhibition of epileptiform activity by inhibiting sodium channels indirectly. (C) 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Role of Nonplanar Peptide Unit in Regular Polypeptide Helices - New Model for Pply-Beta-Benzyl-L-Aspartate

The effect of non-planarity of the peptide unit on helical structures stabilized by intrachain hydrogen bonds is discussed. While the present calculations generally agree with those already reported in the literature for right-handed helical structures, it is found that the most stable left-handed structure is a novel helix, called the delta-helix. Its helical parameters are close to these reported for poly-beta-benzyl-L -aspartate. Conformational energy calculations show that poly-beta-benzyl-L -aspartate with the delta-helical structure is considerably more stable than the structure it is generally believed to take up (the omega-helix) by about 15 kcal/mol-residue.