Site-directed mutagenesis of the conserved threonine (Thr243) of the distal helix of fungal cytochrome P450nor.
Cytochrome P450nor (P450nor) is a heme enzyme which catalyzes NO reduction in denitrifying fungi. Threonine 243 (Thr243) of P450nor, which corresponds to the conserved threonine of monooxygenase cytochrome P450s, was replaced by 18 different amino acids via site-directed mutagenesis. The mutation did not seriously affect the optical absorption and the CD spectral properties of the enzyme in several oxidation, ligation, or spin states or the association rate constant for association of NO with the ferric iron, suggesting subtle and local structural changes in the heme environment on Thr243 mutation. However, the NO reduction activity was dramatically altered by Thr243 mutation, depending on the properties of the replaced amino acids. The catalytic activity, as measured by N2O formation and NADH consumption, was considerably retained on substitution of Asn, Ser, and Gly for Thr243, while it was profoundly decreased or lost on substitution with other amino acids. Kinetic analysis of the reaction of the enzymes with NO and NADH indicated that the decrease in the enzymatic activity upon Thr243 mutation mainly results from a decrease in the rate of reduction of the ferric-NO complex with NADH. On the basis of these enzymatic, kinetic, and spectroscopic results, as well as on the basis of the crystal data for native P450nor [Park, S.-Y., et al. (1997) Nat. Struct. Biol. 4, 827-832], the role of the conserved threonine at the 243 position in the NO reduction reaction by P450nor is discussed. We also discuss structural similarities or differences in the vicinity of the conserved threonine between P450nor and other monooxygenase P450s.
Okamoto
N
N
Department of Veterinary Science, Osaka Prefecture University, Japan.
Imai
Y
Y
Shoun
H
H
Shiro
Y
Y
D002384:Catalysis; D002942:Circular Dichroism; D017124:Conserved Sequence / Q000235:genetics; D018360:Crystallography, X-Ray; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D005670:Fusarium / Q000201:enzymology; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D017433:Protein Structure, Secondary; D013053:Spectrophotometry; D013912:Threonine / Q000235:genetics*
D005656:Fungal Proteins; D013912:Threonine; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; C080610:nitric-oxide reductase (P450)