Role of Thr-252 in cytochrome P450cam: a study with unnatural amino acid mutagenesis.
Replacement of Thr-252 in the active center of cytochrome P450cam with a non-hydroxy amino acid residue such as Ala and Val by conventional site-directed mutagenesis converted this monooxygenase to an NADH oxidase (Imai, M. et al. Proc. Natl. Sci. U. S. A. 86, 7823-7827, 1989). In this study, a mutant enzyme with a methoxy group in place of the hydroxy group of Thr-252 (OMe-mutant) was synthesized by the method of unnatural amino acid mutagenesis (Noren, C. J. et al., Science 244, 182-188, 1989). Unlike other site-directed mutants without a hydroxy group at the position, the OMe-mutant retained a considerably high monooxygenase activity, yielding a stoichiometric amount of 5-exo-hydroxycamphor to that of the oxygen consumed. Thus a free hydroxy group at this position is not an indispensable requisite for the monooxygenase to cleave the O-O bond of molecular O2 as previously proposed.
Kimata
Y
Y
Department of Biochemistry, School of Medicine, Keio University, Tokyo, Japan.
Shimada
H
H
Hirose
T
T
Ishimura
Y
Y
D000409:Alanine; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D019475:Camphor 5-Monooxygenase; D002248:Carbon Monoxide / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D017931:DNA Primers; D006899:Mixed Function Oxygenases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D016958:Pseudomonas putida / Q000201:enzymology*; D012346:RNA, Transfer, Amino Acyl / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry; D013912:Threonine; D014633:Valine
D017931:DNA Primers; D009097:Multienzyme Complexes; D012346:RNA, Transfer, Amino Acyl; D011994:Recombinant Proteins; D013912:Threonine; D002248:Carbon Monoxide; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D019475:Camphor 5-Monooxygenase; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D014633:Valine; D000409:Alanine