EXPRESSION, PURIFICATION AND PRELIMINARY CRISTALOGRAPHIC STUDIES OF CHLOROCATECHOL 1,2-DIOXYGENASE FROM PSEUDOMONAS PUTIDA

BM06

Rustiguel, JKR; Nonato, MC

Laboratório de Cristalografia de Proteínas – Departamento de física e química - Faculdade de Ciências Farmacêuticas de Ribeirão Preto – USP, Ribeirão Preto, S.P., Brazil

The increasing demand of industrialized products and decades of modern agriculture practices are responsible for the increase of organic pollutants discarded in the environment. Aromatic compounds, such as catechol, chlorocatechol and their derivatives, are among the most hazardous ones due to their carcinogenesis potential and recalcitrant properties to degradation. A modern and efficient biotechnological strategy for the elimination of these compounds is called bioremediation and it is based on the use of living microorganisms, or theirs enzymes, to clean up contaminated soil or water. An example, it is the use of dioxygenases, which are bacterial nonheme iron enzymes responsible for the aerobic biodegradation of aromatic compounds. In this work, we present the overexpression, purification and preliminary crystallographic studies of chlorocatechol 1,2-dioxygenase from Pseudomonas putida (Pp 1,2-CCD). This enzyme is a dioxygenase belonging to the intradiol class which shows high specificity to catechol, chlorocatechol and halogenated substrates derived from substituted catechol. Recombinant Pp 1,2-CCD was produced in Escherichia coli BL21(DE3) as a intein-tag fusion protein. Protein expression was induced with 0,5 mM IPTG, for 5 h at 22^oC. Pp 1,2-CCD was purified by affinity chromatography using chitin beads  resin. The protein is eluted with 30 mM of DTT. Following, the enzyme was successfully crystallized using both hanging and sitting drop vapor diffusion methods. Crystals of protein have been obtained using polyethylene glycol 8000 and 10000 as a precipitant agent plus magnesium acetate tetrahydrate as an additive. The brown crystals, with dimensions of 450x80x80 mM, appeared after three days and display a rectangular shape. In order to solve the crystal structure, the crystals will be used for X-ray diffraction experiments at MX-1 beam line located at Laboratório Nacional de Luz Síncrotron. Our studies, corroborated with previous biochemical and biophysical analysis, will be used to fully characterize the enzyme. Our results can be further explored to potentialize the biotechnological use of Pp 1,2-CCD as a bioremediator.