RESUMO
A cowpea class I chitinase (VuChiI) was expressed in the methylotrophic yeast P. pastoris. The recombinant protein was secreted into the culture medium and purified by affinity chromatography on a chitin matrix. The purified chitinase migrated on SDS-polyacrylamide gel electrophoresis as two closely-related bands with apparent molecular masses of 34 and 37 kDa. The identity of these bands as VuChiI was demonstrated by mass spectrometry analysis of tryptic peptides and N-terminal amino acid sequencing. The recombinant chitinase was able to hydrolyze colloidal chitin but did not exhibit enzymatic activity toward synthetic substrates. The highest hydrolytic activity of the cowpea chitinase toward colloidal chitin was observed at pH 5.0. Furthermore, most VuChiI activity (approximately 92%) was retained after heating to 50 °C for 30 min, whereas treatment with 5 mM Cu2+ caused a reduction of 67% in the enzyme's chitinolytic activity. The recombinant protein had antifungal activity as revealed by its ability to inhibit the spore germination and mycelial growth of Penicillium herquei. The three-dimensional structure of VuChiI was resolved at a resolution of 1.55 Å by molecular replacement. The refined model had 245 amino acid residues and 381 water molecules, and the final R-factor and Rfree values were 14.78 and 17.22%, respectively. The catalytic domain of VuChiI adopts an α-helix-rich fold, stabilized by 3 disulfide bridges and possessing a wide catalytic cleft. Analysis of the crystallographic model and molecular docking calculations using chito-oligosaccharides provided evidences about the VuChiI residues involved in sugar binding and catalysis, and a possible mechanism of antifungal action is suggested.
Assuntos
Antifúngicos/metabolismo , Quitinases/metabolismo , Pichia/enzimologia , Proteínas de Plantas/metabolismo , Vigna/enzimologia , Antifúngicos/química , Antifúngicos/farmacologia , Quitinases/química , Quitinases/farmacologia , Hidrólise , Penicillium/efeitos dos fármacos , Proteínas de Plantas/química , Proteínas de Plantas/farmacologia , Ligação ProteicaRESUMO
Global interest in sugarcane has increased significantly in recent years because of its economic impact on sustainable energy production. The purpose of the present study was to evaluate changes in the concentrations of total sugars, amino acids, free proline, and total proteins by colorimetric analyses and nuclear magnetic resonance (NMR) to perform a metabolic profiling of a water-soluble fraction of symplastic sap in response to the constitutive expression of a mutant Δ1-pyrroline-5-carboxylate synthetase (P5CS) gene from Vigna aconitifolia. However, there was not a significant increase in the free proline content in the sap of transgenic plants compared to the non-transformed control plants. The most noticeable difference between the two genotypes was an almost two-fold increase in the accumulation of sucrose in the stem internodes of P5CS transgenic sugarcane plants. The results presented in this work showed that transgenic sugarcane plants with increased levels of free proline accumulates high soluble sugar content and, therefore, may represent a novel genotype for improving sugarcane cultivars.