Cofactor assisted gating mechanism in the active site of NADH oxidase from Thermus thermophilus.
NADH oxidase (NOX) from Thermus thermophilus is a member of a structurally homologous flavoprotein family of nitroreductases and flavin reductases. The importance of local conformational dynamics in the active site of NOX has been recently demonstrated. The enzyme activity was increased by 250% in the presence of 1 M urea with no apparent perturbation of the native structure of the protein. The present in silico results correlate with the in vitro data and suggest the possible explanation about the effect of urea on NOX activity at the molecular level. Both, X-ray structure and molecular dynamics (MD) simulations, show open conformation of the active site represented by approximately 0.9 nm distance between the indole ring of Trp47 and the isoalloxazine ring of FMN412. In this conformation, the substrate molecule can bind in the active site without sterical restraints. MD simulations also indicate more stable conformation of the active site called "closed" conformation. In this conformation, Trp47 and the isoalloxazine ring of FMN412 are so close to each other (approximately 0.5 nm) that the substrate molecule is unable to bind between them without perturbing this conformation. The open/close transition of the active site between Trp47 and the flavin ring is accompanied by release of the "tightly" bound water molecule from the active site--cofactor assisted gating mechanism. The presence of urea in aqueous solutions of NOX prohibits closing of the active site and even unlocks the closed active site because of the concomitant binding of a urea molecule in the active site cavity. The binding of urea in the active site is stabilized by formation of one/two persistent hydrogen bonds involving the carbonyl group of the urea molecule. Our report represents the first MD study of an enzyme from the novel flavoprotein family of nitroreductases and flavin reductases. The common occurrence of aromatic residues covering the active sites in homologous enzymes suggests the possibility of a general gating mechanism and the importance of local dynamics within this flavoprotein family.
Hritz
Jozef
J
Department of Biochemistry, Faculty of Science P. J. Safárik University, Kosice, Slovakia.
Zoldák
Gabriel
G
Sedlák
Erik
E
D001665:Binding Sites; D002942:Circular Dichroism; D003198:Computer Simulation; D006860:Hydrogen Bonding; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000737:chemistry*; D009243:NAD / Q000737:chemistry; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012997:Solvents; D016963:Thermus thermophilus / Q000201:enzymology*; D013997:Time Factors; D014508:Urea / Q000737:chemistry
D009097:Multienzyme Complexes; D012997:Solvents; D009243:NAD; D014508:Urea; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases