Biochemical characterization of aminopeptidases from Xylella fastidiosa and Xanthomonas axonopodis pv citri

Author(s)

Kelly Santos

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2006-12-15

Examining board members

Francisco Javier Medrano; Ana Paula Ulian de Araujo; Ricardo Aparicio; Jose Camillo Novello; Gonçalo Amarante Guimarães Pereira

• Advisor: Francisco Javier Medrano

Abstract

Aminopeptidases release the Nterminal amino acid residue from polypeptides and proteins. They are present in all organisms and play several important roles in food processing, maturation of proteins by elimination of the Nterminal methionine, pathogenicity and many other cellular processes. We report here, the cloning, expression, purification and characterization of a proline iminopeptidase from X.fastidiosa(Xf1510) and a broad specificity aminopeptidase from X.axonopodispv. citri(Xac2987). These two genes have been annotated as putative proline iminopeptidase. Both genes were cloned into the pET15b expression vector, expressed in Escherichia coli, and purified to apparent homogeneity in one step by IMAC. The protein was expressed in the soluble fraction and could be purified in one step by IMAC. Enzymatic assays confirmed Xf1510 as a PIP, and Xac2987 as a broad spectrum aminopeptidase, being able to catalyze the removal of different synthetic substrates. The circular dichroism spectrum allowed us to classify the proteins as part of the a/ß hydrolyses family. Structural studies with pH dependence and thermal stability were preformatted by circular dichroism. The Xf1510 protein presents greater activity in the range of pH between 7,5 and 8,5. The optimum temperature for prolina hydrolysis was 45ºC. The pH optimum for the enzymatic activity of the Xac2987 protein was found in the range of pH of 6,5 and 7,5; being pH optimum 6,6. In this pH the temperature for alanine hydrolysis was found to 40ºC. Structural studies with regard to pH and thermal stability of proteins had been followed by circular dichroism. Studies of thermal and chemical denaturation indicate that the proteins Xf1510 and Xac2987 present intermediate states before reaching the maximum unfolding (AU)