Search

Biotechnological applications of enzymes can involve the use of these molecules under nonphysiological conditions. Thus, it is of interest to understand how environmental variables affect protein structure and dynamics and how this ultimately modulates enzyme function. NADH oxidase (NOX) from Thermus thermophilus exemplifies how enzyme activity can be tuned by reaction conditions, such as temperature, cofactor substitution, and the addition of cosolutes. This enzyme catalyzes the oxidation of reduced NAD(P)H to NAD(P)(+) with the concurrent reduction of O2 to H2O2, with relevance to biosensing applications. It is thermophilic, with an optimum temperature of approximately 65°C and sevenfold lower activity at 25°C. Moderate concentrations (≈1M) of urea and other chaotropes increase NOX activity by up to a factor of 2.5 at room temperature. Furthermore, it is a flavoprotein that accepts either FMN or the much larger FAD as cofactor. We have used nuclear magnetic resonance (NMR) titration and (15)N spin relaxation experiments together with isothermal titration calorimetry to study how NOX structure and dynamics are affected by changes in temperature, the addition of urea and the substitution of the FMN cofactor with FAD. The majority of signals from NOX are quite insensitive to changes in temperature, cosolute addition, and cofactor substitution. However, a small cluster of residues surrounding the active site shows significant changes. These residues are implicated in coupling changes in the solution conditions of the enzyme to changes in catalytic activity.

Enzyme activity is commonly controlled by allostery, where ligand binding at one site alters the activities of distant sites. Classical explanations for multisubunit proteins involve conformational transitions that are fundamentally deterministic. For example, in the Monod-Wyman-Changeaux (MWC) paradigm, conformational transitions occur simultaneously in all subunits. In the Koshland-Nemethy-Filmer (KNF) paradigm, conformational transitions only occur in ligand-bound subunits. In contrast, recent models predict conformational changes that are governed by probabilities rather than absolute rules. To better understand allostery at the molecular level, we applied a recently developed spectroscopic and calorimetric method to the interactions of a dimeric enzyme with two different ligands. We found that conformational transitions appear MWC-like for a ligand that binds at the dimer interface and KNF-like for a distal ligand. These results provide strong experimental support for probabilistic allosteric theory predictions that an enzyme can exhibit a mixture of MWC and KNF character, with the balance partly governed by subunit interface energies.

Biotechnological applications of enzymes can involve the use of these molecules under nonphysiological conditions. Thus, it is of interest to understand how environmental variables affect protein structure and dynamics and how this ultimately modulates enzyme function. NADH oxidase (NOX) from Thermus thermophilus exemplifies how enzyme activity can be tuned by reaction conditions, such as temperature, cofactor substitution, and the addition of cosolutes. This enzyme catalyzes the oxidation of reduced NAD(P)H to NAD(P)(+) with the concurrent reduction of O2 to H2O2, with relevance to biosensing applications. It is thermophilic, with an optimum temperature of approximately 65°C and sevenfold lower activity at 25°C. Moderate concentrations (≈1M) of urea and other chaotropes increase NOX activity by up to a factor of 2.5 at room temperature. Furthermore, it is a flavoprotein that accepts either FMN or the much larger FAD as cofactor. We have used nuclear magnetic resonance (NMR) titration and (15)N spin relaxation experiments together with isothermal titration calorimetry to study how NOX structure and dynamics are affected by changes in temperature, the addition of urea and the substitution of the FMN cofactor with FAD. The majority of signals from NOX are quite insensitive to changes in temperature, cosolute addition, and cofactor substitution. However, a small cluster of residues surrounding the active site shows significant changes. These residues are implicated in coupling changes in the solution conditions of the enzyme to changes in catalytic activity.

We have characterized the backbone dynamics of NADH oxidase from Thermus thermophilus (NOX) using a recently-developed suite of NMR experiments designed to isolate exchange broadening, together with (15)N R (1), R (1ρ ), and {(1)H}-(15)N steady-state NOE relaxation measurements performed at 11.7 and 18.8 T. NOX is a 54 kDa homodimeric enzyme that belongs to a family of structurally homologous flavin reductases and nitroreductases with many potential biotechnology applications. Prior studies have suggested that flexibility is involved in the catalytic mechanism of the enzyme. The active site residue W47 was previously identified as being particularly important, as its level of solvent exposure correlates with enzyme activity, and it was observed to undergo "gating" motions in computer simulations. The NMR data are consistent with these findings. Signals from W47 are dynamically broadened beyond detection and several other residues in the active site have significant R ( ex ) contributions to transverse relaxation rates. In addition, the backbone of S193, whose side chain hydroxyl proton hydrogen bonds directly with the FMN cofactor, exhibits extensive mobility on the ns-ps timescale. We hypothesize that these motions may facilitate structural rearrangements of the active site that allow NOX to accept both FMN and FAD as cofactors.