title: Conformational plasticity surrounding the active site of NADH oxidase from Thermus thermophilus.
abstract: 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.
authors.affiliation: Department of Chemistry, McGill University, Montreal, Quebec, H3A 0B8.
authors.forename: Teresa
authors.initials: T
authors.lastname: Miletti
authors.affiliation: Department of Chemistry, McGill University, Montreal, Quebec, H3A 0B8.
authors.forename: Justin
authors.initials: J
authors.lastname: Di Trani
authors.affiliation: Laboratoire de biologie moléculaire, Département des Sciences Biologiques, Centre BioMed, Université du Québec à Montréal, Montréal, Québec, H3C 3P8.
authors.forename: Louis-Charles
authors.initials: LC
authors.lastname: Levros
authors.affiliation: Department of Chemistry, McGill University, Montreal, Quebec, H3A 0B8.
authors.forename: Anthony
authors.initials: A
authors.lastname: Mittermaier
mesh_terms: D001665:Binding Sites; D020134:Catalytic Domain; D005486:Flavin Mononucleotide / Q000737:chemistry / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D011487:Protein Conformation; D013696:Temperature; D016963:Thermus thermophilus / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D014508:Urea / Q000378:metabolism*
chemical_list: D009097:Multienzyme Complexes; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; D014508:Urea; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases
keywords: NMR titration; enzyme cofactor substitution; isothermal titration calorimetry; nuclear magnetic resonance; spin relaxation; thermophile; urea activation