Aromatic stacking as a determinant of the thermal stability of CYP119 from Sulfolobus solfataricus.
Two notable features of the thermophilic CYP119, an Arg154-Glu212 salt bridge between the F-G loop and the I helix and an extended aromatic cluster, were studied to determine their contributions to the thermal stability of the enzyme. Site-specific mutants of the salt bridge (Arg154, Glu212) and aromatic cluster (Tyr2, Trp4, Trp231, Tyr250, Trp281) were expressed and purified. The substrate-binding and kinetic constants for lauric acid hydroxylation are little affected in most mutants, but the E212D mutant is inactive and the R154Q mutant has higher K(s),K(m), and k(cat) values. The salt bridge mutants, like wild-type CYP119, melt at 91+/-1 degrees C, whereas mutation of individual residues in the extended aromatic cluster lowers the T(m) by 10-15 degrees C even though no change is observed on mutation of an unrelated aromatic residue. The extended aromatic cluster, but not the Arg154-Glu212 salt bridge, contributes to the thermal stability of CYP119.
Puchkaev
Andrei V
AV
Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco 94143-0446, USA.
Koo
Laura S
LS
Ortiz de Montellano
Paul R
PR
D019843:Archaeal Proteins; D002942:Circular Dichroism; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D005288:Ferredoxins / Q000737:chemistry; D006358:Hot Temperature; D006900:Hydroxylation; D007700:Kinetics; D007850:Lauric Acids / Q000737:chemistry / Q000378:metabolism; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry; D010105:Oxygenases / Q000737:chemistry*; D011485:Protein Binding; D011489:Protein Denaturation; D012492:Salts / Q000737:chemistry; D013379:Substrate Specificity; D016944:Sulfolobus / Q000201:enzymology*; D013696:Temperature; D014466:Ultraviolet Rays
D019843:Archaeal Proteins; D005288:Ferredoxins; D007850:Lauric Acids; D012492:Salts; C030358:lauric acid; C100137:putidaredoxin; D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases; C408874:CYP119 protein, Sulfolobus solfataricus; D009247:NADH, NADPH Oxidoreductases; C022486:putidaredoxin reductase