Phosphoenolpyruvate carboxykinase from T. cruzi magnetic beads affinity-based screening assays on crude plant extracts from Brazilian Cerrado.

In T. cruzi, a causative agent of Chagas disease, phosphoenolpyruvate carboxykinase (TcPEPCK) is associated with carbohydrate catabolism. Due to its importance in the metabolism of the parasite, it has become a promising target for the development of new drugs against Chagas disease. Aiming to investigate different approaches for ligands screening, TcPEPCK was immobilized on amine-terminated magnetic beads (TcPEPCK-MB) and kinetically characterized by liquid chromatography tandem mass spectrometry activity assay with a KMapp value of 10 ± 1 µM to oxaloacetate as substrate. Natural products library affords highly diverse molecular frameworks through their secondary metabolites, herein a ligand fishing TcPEPCK-MB assay is described for prospecting ligands in four ethanolic extracts of Brazilian Cerrado plants: Qualea grandiflora (Vochysiaceae), Diospyros burchellii (Ebenaceae), Anadenanthera falcata (Fabaceae) and Byrsonima coccolobifolia (Malpighiaceae). The chemical characterization of eleven identified ligands was carried out by liquid chromatography tandem high-resolution mass spectrometry experiments. Senecic acid, syneilesinolide A, phytosphingosine and vanillic acid 4-glucopyranoside are herein reported for the first time for Q. grandiflora, D. burchellii, A. falcata, respectively. In addition, the specificity of the assay was observed since only catechin was fished out from the ethanolic extract of B. coccolobifolia leaves, despite the presence of epicatechin epimer.

Inhibition of Leishmania amazonensis arginase by fucogalactan isolated from Agrocybe aegerita mushroom.

The inhibition of arginase from Leishmania spp. is considered a promising approach to the leishmaniasis treatment. In this study, the potential of a fucogalactan isolated from the medicinal mushroom Agrocybe aegerita was evaluated against arginase (ARG) from Leishmania amazonensis. The polysaccharide was obtained via aqueous extraction, and purified by freeze thawing and precipitation with Fehling solution. Its chemical structure was established by monosaccharide composition, methylation analysis, partial acid hydrolysis, and NMR spectroscopy. The data indicated that it is a fucogalactan (FG-Aa; Mw = 13.8 kDa), having a (1→6)-linked α-D-Galp main-chain partially substituted in O-2 by non-reducing end-units of α-L-Fucp. FG-Aa showed significant inhibitory activity on ARG with IC50potency of 5.82 ± 0.57 µM. The mechanism of ARG inhibition by the heterogalactan was the competitive type, with Kiof 1.54 ± 0.15 µM. This is the first report of an inhibitory activity of arginase from L. amazonensis by biopolymers, which encourages us to investigate further polysaccharides as a new class of ARG inhibitors.

A molecular mechanism for Lys49-phospholipase A2 activity based on ligand-induced conformational change.

Agkistrodon contortrix laticinctus myotoxin is a Lys(49)-phospholipase A(2) (EC 3.1.1.4) isolated from the venom of the serpent A. contortrix laticinctus (broad-banded copperhead). We present here three monomeric crystal structures of the myotoxin, obtained under different crystallization conditions. The three forms present notable structural differences and reveal that the presence of a ligand in the active site (naturally presumed to be a fatty acid) induces the exposure of a hydrophobic surface (the hydrophobic knuckle) toward the C terminus. The knuckle in A. contortrix laticinctus myotoxin involves the side chains of Phe(121) and Phe(124) and is a consequence of the formation of a canonical structure for the main chain within the region of residues 118-125. Comparison with other Lys(49)-phospholipase A(2) myotoxins shows that although the knuckle is a generic structural motif common to all members of the family, it is not readily recognizable by simple sequence analyses. An activation mechanism is proposed that relates fatty acid retention at the active site to conformational changes within the C-terminal region, a part of the molecule that has long been associated with Ca(2+)-independent membrane damaging activity and myotoxicity. This provides, for the first time, a direct structural connection between the phospholipase "active site" and the C-terminal "myotoxic site," justifying the otherwise enigmatic conservation of the residues of the former in supposedly catalytically inactive molecules.

Overexpression, purification, and biochemical characterization of GumC, an enzyme involved in the biosynthesis of exopolysaccharide by Xylella fastidiosa.

GumC is one of nine enzymes involved in the biosynthesis of fastidian gum, an exopolysaccharide produced by Xylella fastidiosa that may be linked directly to the pathogenicity of the microorganism. GumC may be responsible for gum polymerization or secretion through the membrane of X. fastidiosa. To perform structure and functions studies, we developed an expression system for the production of GumC as a fusion protein with maltose binding protein (MBP) using pMAL-c2x vector. The GumC-MBP fusion protein was expressed as a 94 kDa protein, which strongly reacts with anti-MBP antibodies. GumC-MBP was isolated by affinity chromatography through an amylose column and used to produce antibodies against the fusion protein. After the enzymatic cleavage of MBP, GumC was purified on a Q Sepharose Fast Flow column. GumC showed a molecular weight corresponding to the expected one (52 kDa) and its N-terminal sequence was identical to that deduced from the DNA. The shape of the circular dichroism spectrum was compatible with a folded protein that contains alpha-helical regions in its structure. Therefore, in this study we describe, for the first time, the production of GumC recombinant protein.