Fungal P450nor: expression in Escherichia coli and site-directed mutageneses at the putative distal region.
P450nor, nitric oxide reductase from Fusarium oxysporum, was expressed in the soluble fraction of Escherichia coli cells by modifying the N-terminal codons and utilizing the pCW vector. The modified P450nor purified to electrophoretic homogeneity had spectral and enzymatic properties identical to those of native P450nor obtained from the fungi. Residues 239 to 247 of the modified P450nor were replaced with Lys by site-directed mutagenesis. Thr-243 to His- and Arg-mutants were also created. Among 11 mutants, only the Thr-243 to Lys-mutant exhibited an absorption spectrum characteristic of a nitrogenous ligand-bound form of P450 at pH 8.0 in the ferric state, but the spectrum was altered to that of the wild-type P450nor as the pH was lowered. Other mutants had spectra typical of the low- and high-spin mixed form of P450 in the ferric state. In the ferrous state, all mutants showed the same spectrum as the wild-type P450nor. Nitric oxide reductase activity was considerably decreased by the replacement of Thr-243 with Lys, His, or Arg or Ala-239 with Lys. These findings indicate that Thr-243 is located more closely to the heme iron than other residues in the putative distal helix of P450nor and plays an important role in the catalytic activity, but a specific difference in the structure of the heme pocket from other P450s is suggested.
Okamoto
N
N
Department of Veterinary Science, Osaka Prefecture University, Sakai, Japan.
Tsuruta
K
K
Imai
Y
Y
Tomura
D
D
Shoun
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005670:Fusarium / Q000201:enzymology*; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013057:Spectrum Analysis; D013912:Threonine / Q000737:chemistry; D014169:Transformation, Bacterial
D018076:DNA, Complementary; D011994:Recombinant Proteins; D009243:NAD; D013912:Threonine; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; C080610:nitric-oxide reductase (P450)