Structure of a calcium-independent phospholipase-like myotoxic protein from Bothrops asper venom

Myotoxin II, a myotoxic calcium-independent phospholipase-like protein isolated from the venom of Bothrops asper, possesses no detectable phospholipase activity. The crystal structure has been determined and refined at 2.8 Angstrom to an R factor of 16.5% (F>3 sigma) with excellent stereochemistry. Amino-acid differences between catalytically active phospholipases and myotoxin LI in the Ca2+-binding region, specifically the substitutions Tyr28-->Asn, Gly32-->Leu and Asp49-->Lys, result in an altered local conformation. The key difference is that the epsilon-amino group of Lys49 fills the site normally occupied by the calcium ion in catalytically active phospholipases. In contrast to the homologous monomeric Lys49 variant from Agkistrodon piscivorus piscivorus, myotoxin II is present as a dimer both in solution and in the crystalline state. The two molecules in the asymmetric unit are related by a nearly perfect twofold axis, yet the dimer is radically different from the dimer formed by the phospholipase from Crotalus atrox. Whereas in C. atrox the dimer interface occludes the active sites, in myotoxin II they are exposed to solvent.

Molecular models of cyclin-dependent kinase 1 complexed with inhibitors

Roscovitine and flavopiridol have been shown to potently inhibit cyclin-dependent kinase 1 and 2 (CDK1 and 2). The structures of CDK2 complexed with roscovitine and deschoroflavopiridol have been reported, however no crystallographic structure is available for complexes of CDK1 with inhibitors. The present work describes two molecular models for the binary complexes CDK1:roscovitine and CDK1:flavopiridol. These structural models indicate that both inhibitors strongly bind to the ATP-binding pocket of CDKI and structural comparison of the CDK complexes correlates the structures with differences in inhibition of these CDKs by flavopiridol and roscovitine. This article explains the structural basis for the observed differences in activity of these inhibitors. (C) 2004 Elsevier B.V. All rights reserved.

A 4.2 kDa synthetic peptide as a potential probe to evaluate the antibacterial activity of coumarin drugs

The coumarin antibiotics are potent inhibitors of DNA replication whose target is the enzyme DNA gyrase, an ATP-dependent bacterial type II topoisomerase. The coumarin drugs inhibit gyrase action by competitive binding to the ATP-binding site of DNA gyrase B protein. The production of new biologically active products has stimulated additional studies on coumarin-gyrase interactions. In this regard, a 4.2 kDa peptide mimic of DNA gyrase B protein from Escherichia coli has been designed and synthesized. The peptide sequence includes the natural fragment 131-146 (coumarin resistance-determining region) and a segment containing the gyrase-DNA interaction region (positions 753-770). The peptide mimic binds to novobiocin (K-a = 1.4 +/- 0.3 x 10(5) m(-1)), plasmid (K-a = 1.6 +/- 0.5 x 10(6) m(-1)) and ATP (K-a = 1.9 f 0.4 x 10(3) m(-1)), results previously found with the intact B protein. on the other hand, the binding to novobiocin was reduced when a mutation of Arg-136 to Leu-136 was introduced, a change previously found in the DNA gyrase B protein from several coumarin-resistant clinical isolates of Escherichia coLi. In contrast, the binding to plasmid and to ATP was not altered. These results suggest that synthetic peptides designed in a similar way to that described here could be used as mimics of DNA gyrase in studies which seek a better understanding of the ATP, as well as coumarin, binding to the gyrase and also the mechanism of action of this class of antibacterial drugs. Copyright (C) 2004 European Peptide Society and John Wiley Sons, Ltd.

CRYSTALLIZATION AND PRELIMINARY DIFFRACTION DATA OF 2 MYOTOXINS ISOLATED FROM THE VENOMS OF BOTHROPS-ASPER (TERCIOPELO) AND BOTHROPS-NUMMIFER (JUMPING VIPER)

Two myotoxins isolated from B. asper (myotoxin II) and B. nummifer (myotoxin I) snake venoms have been crystallized and their diffraction properties are described. These myotoxins are phospholipase A2 variants which lack enzymatic activity; B. asper myotoxin II is a lysine-49 phospholipase. Crystals were obtained at room temperature by standard hanging-drop vapour diffusion methods. Crystals diffracted to a resolution of 2.8 and 2.3 angstrom, respectively.

Indole glucoalkaloids from Chimarrhis turbinata and their evaluation as antioxidant agents and acetylcholinesterase inhibitors

As part of our study on bioactive agents from Brazilian rainforest plants, two new glucoalkaloids, 3,4-dehydro-strictosidine (1) and 3,4-dehydro-strictosidinic acid (2), were isolated from Chimarrhis turbinata, along with seven known glucoalkaloids, cordifoline (3), strictosidinic acid (4), strictosidine (5), 5alpha-carboxystrictosidine (6), turbinatine (7), desoxycordifoline (8), and harman-3-carboxylic acid (9). The structures of the new alkaloids were established on the basis of comprehensive spectral analysis, mainly 1D and 2D NMR experiments, as well as high-resolution HRESIMS. Alkaloid 3 showed strong free-radical scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) as well as pronounced antioxidant activity evidenced by redox properties measured by ElCD-HPLC. Additionally, alkaloids 1-9 were submitted to TLC screening for acetylcholinesterase inhibitors. Both 7 and 8 were shown to be moderate acetylcholinesterase inhibitors at a concentration of 0.1 and 1.0 muM, respectively. In an in vitro rat brain assay, 7 showed moderate activity (IC50 1.86 muM), compared to the standard compound, galanthamine (IC50 0.92 muM).

OSSEOUS PLATE ALKALINE-PHOSPHATASE IS ANCHORED BY GPI

Alkaline phosphatase activity was released up to 100% from the membrane by using 0.1 U of phosphatidylinositol-specific phospholipase C from B. thuringiensis. The Mr of solubilized enzyme was 145,000 by Sephacryl S-300 gel filtration and 66,000 by SDS-PAGE, suggesting a dimeric structure. Solubilization of the membrane-bound enzyme with phospholipase C did not destroy its ability to hydrolyze p-nitrophenyl phosphate (PNPP) (264.3 mu mol min(-1) mg(-1)), ATP (42.0 mu mol min(-1) mg(-1)) and pyrophosphate (28.4 mu mol min(-1) mg(-1)). The hydrolysis of ATP and PNPP by solubilized enzyme exhibited ''Michaelian'' kinetics with K-0.5 = 70 and 979 mu M, respectively. For pyrophosphate, K-0.5 was 128 mu M and site-site interactions were observed (n = 1.4). Magnesium ions were stimulatory (K-d = 1.5 mM) but zinc ions were powerful non-competitive inhibitors (K-d = 6.2 mu M) of solubilized enzyme. Treatment of solubilized alkaline phosphatase with Chellex 100 reduced the original PNPPase activity to 5%. Cobalt (K-0.5 = 10.1 mu M), magnesium (K-0.5 = 29.5 mu M) and manganese ions (K-0.5 = 5 mu M) restored the activity of the apoenzyme with positive cooperativity, suggesting that phosphatidylinositol-specific phospholipase C-solubilized alkaline phosphatase is a metalloenzyme. The stimulation of the apoenzyme by calcium ions (K-0.5 = 653 mu M) was lower than that observed for the other ions (26%) and exhibited site-site interactions (n = 0.7). Zinc ions had no effect on the apoenzyme of the solubilized enzyme.

4 '-Aminochalcones As Novel Inhibitors of the Chlorinating Activity of Myeloperoxidase

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Natural Products from Garcinia brasiliensis as Leishmania Protease Inhibitors

The infections by protozoans of the genus Leishmania are a major worldwide health problem, with high endemicity in developing countries. The drugs of choice for the treatment of leishmaniasis are the pentavalent antimonials, which cause renal and cardiac toxicity. As part of a search for new drugs against leishmaniasis, we evaluated the in vitro Leishmania protease inhibition activity of extracts (hexanic, ethyl-acetate, and ethanolic) and fukugetin, a bioflavonoid purified from the ethyl-acetate extract of the pericarp of the fruit of Garcinia brasiliensis, a tree native to Brazilian forests. The isolated compound was characterized by using spectral analyses with nuclear magnetic resonance, mass spectroscopy, ultraviolet, and infrared techniques. The ethyl-acetate extract and the compound fukugetin showed significant activity as inhibitors of Leishmania's proteases, with mean (+/- SD) IC(50) (50% inhibition concentration of protease activity) values of 15.0 +/- 1.3 mu g/mL and 3.2 +/- 0.5 mu M/mL, respectively, characterizing a bioguided assay. In addition, this isolated compound showed no activity against promastigote and amastigote forms of L. (L.) amazonensis and mammalian cells. These results suggest that fukugetin is a potent protease inhibitor of L. (L.) amazonensis and does not cause toxicity in mammalian or Leishmania cells in vitro. This study provides new perspectives on the development of novel drugs that have leishmanicidal activity obtained from natural products and that target the parasite's proteases.

Structure and catalytic mechanism of glucosamine 6-phosphate deaminase from Escherichia coli at 2.1 Å resolution

Background: Glucosamine 6-phosphate deaminase from Escherichia coli is an allosteric hexameric enzyme which catalyzes the reversible conversion of D-glucosamine 6-phosphate into D-fructose 6-phosphate and ammonium ion and is activated by N-acetyl-D-glucosamine 6-phosphate. Mechanistically, it belongs to the group of aldose-ketose isomerases, but its reaction also accomplishes a simultaneous amination/deamination. The determination of the structure of this protein provides fundamental knowledge for understanding its mode of action and the nature of allosteric conformational changes that regulate its function. Results: The crystal structure of glucosamine 6-phosphate deaminase with bound phosphate ions is presented at 2.1 Å resolution together with the refined structures of the enzyme in complexes with its allosteric activator and with a competitive inhibitor. The protein fold can be described as a modified NAD-binding domain. Conclusions: From the similarities between the three presented structures, it is concluded that these represent the enzymatically active R state conformer. A mechanism for the deaminase reaction is proposed. It comprises steps to open the pyranose ring of the substrate and a sequence of general base-catalyzed reactions to bring about isomerization and deamination, with Asp72 playing a key role as a proton exchanger.

Heat treatment and inactivation of trypsin-chymotrypsin inhibitors and lectins from beans (Phaseolus vulgaris L.)

This work investigates some factors affecting the inactivation of common bean trypsin inhibitor and phytohemagglutin. Trypsin inhibitor activity was totally stable to heat treatment (30 min, 97C) in the total protein extract, albumin or globulin fraction. Heat treatment of the whole beans easily inactivated the inhibitor. Heat resistance of trypsin inhibitor was intermediate in the bean flour which received the same heat treatment. Independent of sample, the inhibitor was very stable to heat treatment at neutral and acidic pH and labile under strong alkaline conditions. Heating for 30 min in boiling water at pH 12 resulted in complete inactivation of the trypsin inhibitor. Autoclaving (121C) soaked whole beans and flour for 5 min inactivated 55% of the trypsin inhibitor activity in the soaked flour and 75% in the whole beans. After autoclaving 20 min, inactivation of trypsin inhibitor was about 65% in the flour and 80% in the whole beans. The phytohemagglutinin (lectin) activity was totally destroyed in the autoclaved beans after 5 min and in the flour after 15 min.