Aspectos físico-químicos sobre a reativação e a estabilidade da 1,2-dihidroxinaftaleno dioxigenase (DoxG)

Abstract

Bacteria of the genus Pseudomonas have a formidable arsenal of enzymes allowing them to use polycyclic aromatic hydrocarbons (PAHs) as the sole carbon source. Among these enzymes, the 1,2-dihydroxynaphthalene dioxygenase from Pseudomonas sp. C18 (DoxG), a Fe+2-dependent dioxygenase extradiol, catalyzes the conversion of 1,2-dihydroxynaphthalene to 2-hydroxy-2H-chromene-2-carboxylate via a dioxygenation. A reaction with no match in classical synthesis. This enzyme exhibits a large active site endorsing its promiscuity, which is able to cleave, in addition to a series of catechols, the polychlorinated biphenyls (BPCs). This formidable capacity contrasts with the facile oxidation of the Fe+2 cofactor by the oxygen present in the air, a process that occurs throughout the lysis and purification of the enzyme and leads to its inactivation. Our goal is to determine the factors to efficient enzyme reactivation, as well as strategies to improve its stability and catalytic activity. Therefore, DoxG was expressed and purified under aerobic conditions. Characterization was done by its monomeric mass determined by electrophoresis and by its oligomeric state estimated using dynamic light scattering, which was consistent with an octamer in aqueous solution showing a molecular mass of about 270 kDa. The rate of holoenzyme reconstitution was sensitive to several factors: (1) incubation with Fe2+ was the most efficient form of reactivation; (2) the reactivation rate depends on concentration of the Fe2+; (3) the pH of 5.75 provided the fastest reactivation. In more alkaline media the availability of the cofactor was limited and in more acidic media the process was negatively affected by protonation of active-site residues involved in the complexation of the cofactor; (4) although the ionic strength using NaCl or KCl did not affect the reactivation, salts or buffers with anions capable of binding Fe+2 (e.g. sulfate) affected its availability; (5) reactivation of 90% of the enzyme required two and a half hours of incubation with Fe2+. In addition, it was observed that Mn+2 could not replace the Fe+2 as cofactor, however, it provided greater stability to DoxG. Thermal inactivation studies showed that in the presence of Mn+2, the enzyme did not lose its activity considerably after being incubated for forty minutes at 60 °C. The enzyme activity studies showed that DoxG was able to catalyze the cleavage of catechol, 3-methylcatechol, and 2,3-dihydroxybiphenyl. The catalytic efficiency (kcat/Km) for cleavage of 2,3-dihydroxybiphenyl was 1000-fold and 10-fold greater than catechol and 3-methylcatechol, respectively.