Engineering of a simplified nitrate reductase and study of the dynamic catalysis of nitrate reductase and its catalytic fragments

Nitrate reductase (NaR) is the first enzyme involved in the N-assimilation metabolism. It catalyses the reduction of nitrate to nitrite by coupling it with the oxidation of NADH, NADPH, or both in the case of bispecific NaR. The enzyme has two active sites: the NAD(P)H electron donor site and the nitrate reducing site. It was shown to be a homo-dimer with each monomer composed of 900 amino acids. NaR monomer has a Mw of 100 kDa, and the monomer contains one of each of the following cofactors: flavin adenine nucleotide (FAD), heme-Fe and molybdenum-molybdopterin (Mo-MPT). The cofactors are present in a ratio of 1:1:1, and they act like an internal electron transport chain, with a downhill redox potential from NAD(P)H to nitrate. NaR is organized in three functional domains: FAD domain which contains the nucleotide binding site and the FAD binding site, the Cytochrome b (Cyt b) domain which binds the heme-Fe, and the Mo-MPT domain, which contains the dimer interface, the Mo-MPT binding domain and the nitrate binding domain.; NaR previously was purified from its original source, but the availability on the market of an expression system able to produce large amounts of active holo-NaR has allowed the in depth study of NaR, permitting researchers to engineer the enzyme. Some of the NaR functional domains have been independently expressed in the methylotrophic yeast Pichia pastoris, and have shown partial activities of similar values to the one found in the holo-enzyme. In this study, a simplified nitrate reductase was engineered from a yeast nitrate reductase. It contained the Mo-MPT functional domain, and it was expressed in large amounts by fermentation in Pichia. It showed a specific nitrate reductase activity, making it the simplest form of the enzyme capable of nitrate reduction. Biochemical characterizations showed that the simplified form was behaving like the Mo-MPT domain of the holo-enzyme.; Recent studies have shown that the limiting events of NaR catalysis involved the Cyt b domain. Viscosity studies were performed to try to determine the nature of this limitation by testing some NaR catalytic fragment and some holo-NaR with partial (ferricyanate reductase activity) and total enzymatic assay (NaR activity) at different viscosities. The ferricyanate (FeCN) reductase activity involves the FAD and the Cyt b domain, and the NaR activity involves the totality of the electron transport chain of the enzyme. Analysis showed that the kcatobs were decreasing when the viscosity was increased. The limiting factor seemed to be a translational motion occurring during the catalytic turnover of the enzyme. It was found that the compositions of the hinges surrounding the Cyt b domain seemed to affect the Cyt b translational movement, and therefore the viscosity impact on the enzyme. A graphical method was developed based on dynamic model equations of the NaR catalysis in order to calculate the work of the "drag force" (Wd) of the cytochrome b domain on catalysis. According to these results, the drag work of the Cyt b during catalytic turnover was almost tripled in the NaR activity, suggesting that the translational movement of the Cyt b was the limiting factor of the catalysis. Furthermore, the results showed that the Wd varied with the length of the Hinge 2. The equations derived may be useful for predicting the effect of viscosity on enzymes having a large conformational change during catalysis.