Função de resíduos do sítio de ligação do FAD da salicilato hidroxilase sobre a sua capacidade de ligação, catálise e formação de produtos

Abstract

Salicylate hydroxylase (NahG) from Pseudomonas putida is a FAD-dependent flavoenzyme of the naphthalene degradation pathway that catalyzes the oxidative decarboxylation of salicylate into catechol, carbon dioxide and water, with consumption of NADH and molecular oxygen. Catalysis involves a flavin-hydroperoxide intermediate of FAD, which, together with a conserved histidine residue (H226) plays a central role in salicylate conversion. Other conserved residues near to the FAD and salicylate binding sites are E38, D314 and R111. Herein, the thermodynamic and kinetic effects of site-directed mutations E38A, D314A and R111Q were evaluated in relation to FAD binding capacity and catalysis of the salicylate oxidative decarboxylation. The binding Gibbs free energies for FAD were determined from the dissociation constants (Kd) of the E.FAD complexes determined using fluorometric titrations. The Kd value of 0.036 μM for the native NahG corresponds to a ΔG° of -10.2 kcal mol-1 at 25 ºC, which is not altered by the R111Q mutation (Kd = 0.034 μM), suggesting that this residue has not an apparent role in FAD binding in contrast to structural observations that suggest strong hydrogen bonds between residue R111 and the phosphoryl group of FAD. The E38A and D314A mutations result in higher Kd values, 8.94 and 4.71 μM, respectively, which correspond to weaker E.FAD complexes by 3.3 and 2.9 kcal mol-1, respectively, when compared to the native enzyme. These results suggest that residues E38 and D314 together contribute with 6.2 kcal mol-1, approximately 60% of the total binding energy of FAD in the native enzyme. In the kinetic studies of the mutants, a significant effect of approximately 1800 times on kcat was observed due to the R111Q mutation and a total shift in the partitioning of the flavohydroperoxide species to the uncoupled pathway that leads to the production of H2O2 without any conversion of the substrate (coupled pathway). Suggesting that residue R111 may favor an open conformation, where the isoalloxazine group of FAD is active for reduction by NADH and oxygenation, but unable to produce substrate conversion, which requires a closed conformation according to structural data. The joint effect of 1.6 kcal mol-1 at 25 °C of residues E38 and D314 represents about 10% of the Gibbs free energy of activation for kcat. These effects partially reflect on the enzyme's ability to stabilize the flavohydroperoxide species and its capacity to carry out substrate conversion. Finally, to determine the proportion between the uncoupled and coupled pathways, a new kinetic method was developed, which has the advantage over colorimetric methods of being able to determine the proportion of pathways within the first 10% of NADH oxidation.