用细胞色素P-450（睾酮16α-羟化酶）的羧基末端28个残基替换兔肝细胞色素P-450（月桂酸（ω-1）羟化酶）的相应残基，可产生一种新的立体特异性羟化酶活性。
Replacing the carboxy-terminal 28 residues of rabbit liver P-450 (laurate (omega-1)-hydroxylase) with those of P-450 (testosterone 16 alpha-hydroxylase) produces a new stereospecific hydroxylase activity.
作者信息
Uno T, Yokota H, Imai Y
机构信息
Institute for Protein Research, Osaka University, Japan.
出版信息
Biochem Biophys Res Commun. 1990 Mar 16;167(2):498-503. doi: 10.1016/0006-291x(90)92051-z.
cDNA for chimeric P-450 consisting of the amino-terminal 462 residues of P-450 (laurate (omega-1)-hydroxylase) and the remaining 28 residues of P-450 (testosterone 16 alpha-hydroxylase) was constructed and expressed in yeast cells. The resulting chimera could catalyze laurate (omega-1)-hydroxylation and benzphetamine N-demethylation at much higher rates than the parental P-450s, but exhibited the same specificity towards fatty acid substrates as the wild-type laurate hydroxylase. When testosterone was examined as a substrate, the 16 beta-hydroxylated product, which cannot be formed by either of the parental P-450s, was detected, suggesting that the laurate hydroxylase contains a structure that is capable of binding testosterone at a proper orientation so that it can be hydroxylated at the 16 beta position.
构建了由P-450（月桂酸（ω-1）羟化酶）的氨基末端462个残基和P-450（睾酮16α-羟化酶）的其余28个残基组成的嵌合P-450的cDNA，并在酵母细胞中进行表达。所得嵌合体催化月桂酸（ω-1）羟化和苄非他明N-去甲基化的速率比亲本P-450高得多，但对脂肪酸底物的特异性与野生型月桂酸羟化酶相同。当检测睾酮作为底物时，检测到亲本P-450均无法形成的16β-羟基化产物，这表明月桂酸羟化酶含有一种能够以适当方向结合睾酮的结构，从而使其能够在16β位进行羟化。
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