“pH effects on the reaction catalyzed by Aspergillus fumigatus Siderophore A (AfSidA)”
Instituto Tecnológico de Costa Rica. Escuela de Biología.
Fralin Biotechnology Center, Virginia Tech.
Proyecto de Graduación (Bachillerato en Ingeniería en Biotecnología). Instituto Tecnológico de Costa Rica. Escuela de Biología, 2011.
Aspergilli is the name given to a family of saprophytic and ubiquitous fungus, that are important as industrial microorganisms, responsible of the large-scale production of several metabolites and enzymes. From this genus, Aspergillus fumigatus is one of the most important, because it is known as the leading mold pathogen in immunocompromised patients, causing aspergillosis, which is an infection that affects the respiratory system. Several studies revealed that one crucial step of the infection is the acquisition of nutrients, like iron, which availability in the human serum is really limited. For that reason, A. fumigatus biosynthesizes siderophores, to overcome the problem of limited iron accessible. For the synthesis of these molecules, A. fumigatus use an enzyme called N5-ornithine hydroxylase (AfSidA), which catalyzes the first committed step in the synthesis. The information of the enzyme is really limited, however the siderophore system have attracted much attention, so the enzyme, which represents a possible target for fungal therapies, that is why biochemical analysis are important. The principal objective was the evaluation in vitro of the enzymatic activity of the ornithine hydroxylase, by varying the concentration of selected compounds and pH. For these evaluations, AfSidA was first, expressed and purified; then according to the activity and stability of the enzyme in a series of buffers, a buffer system was selected to performed oxygen consumption assays, using constant concentrations of coenzyme, NADH 1mM, and enzyme, 2uM; and varying the concentrations of the selected substrates, L-ornithine and L-lysine. Also an analysis of a possible effect caused by NADH was done. The results obtained in both profiles using the substrates were very similar, obtaining for the kcat profile one pK related to the formation of an active species, while in both kcat/KM profiles, two pKs were presented, the lower one representing the formation of an active species and the higher one, the transition to a less active species. In the case of the NADH profile, no uncoupling effect was observed, and tow ionizable groups were identified. The pKs resultants form the analysis can be highly related to a deprotonation on the substrates, leading to the formation of active specie, or the activation of the enzyme, when it reacts with NADH; and the protonation pKs can be linked to the formation of the protonated intermediate, C4a-peroxyflan.