The omega-hydroxylation of lauric acid: Oxidation of 12-hydroxylauric acid to dodecanedioic acid by a purified recombinant fusion protein containing P450 4A1 and NADPH-P450 reductase
The omega-hydroxylation of lauric acid: Oxidation of 12-hydroxylauric acid to dodecanedioic acid by a purified recombinant fusion protein containing P450 4A1 and NADPH-P450 reductase
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DOI：
10.1006/abbi.1996.0243
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发表时间：
1996-06-01
影响因子：
3.9
通讯作者：
Estabrook, RW
中科院分区：
文献类型：
--
作者：
Shet, MS;Fisher, CW;Estabrook, RW
重组融合蛋白rF450[mRat4A1/mRatOR]L1含有P450 4A1的血红素结构域和NADPH-P450还原酶的黄素结构域，当与二脲酰磷脂酰胆碱（DLPC）、Chaps、细胞色素b(5)和20倍过量的纯化NADPH-P450还原酶一起培养时，以约300 nmol/min/nmol P450的速率催化月季酸的ω -氧化。这是首次报道具有如此高周转率的哺乳动物P450酶。得到的12-羟基十二烷酸[12-羟基月桂酸（12- oh LA）]经12,12-二羟基十二烷酸，P450加氧酶进一步氧化为十二烷二酸（癸烷-1,10-二羧酸）。光谱结合研究表明，12-OH LA抑制月季酸与P450活性位点的结合，K-i约为1.9 μ m。描述了重组P450 4A1的构建和表达，重组P450 4A1、DLPC、Chaps、b(5)和纯化的NADPH-P450还原酶的重组实验结果与纯化的融合蛋白相似。尽管流动率较低。采用正相高效液相色谱法测定月桂酸代谢过程中产生的代谢产物。(C) 1996学术出版社，Inc.
The recombinant fusion protein rF450[mRat4A1/mRatOR]L1, containing the heme domain of P450 4A1 and the flavin domains of NADPH-P450 reductase, when incubated with dilaurylphosphatidylcholine (DLPC), Chaps, cytochrome b(5), and a 20-fold excess of purified NADPH-P450 reductase, catalyzes the omega-oxidation of lauric acid at a rate of about 300 nmol/min/nmol P450. This is the first report of a mammalian P450 enzyme with such a high turnover number. The resultant 12-hydroxydodecanoic acid [12-hydroxylauric acid (12-OH LA)] is further oxidized by the P450 oxygenase reaction to dodecanedioic acid (decane-1,10-dicarboxylic acid) via 12,12-dihydroxydodecanoic acid. Spectral binding studies show that 12-OH LA inhibits the binding of lauric acid to the active site of P450 with a K-i of about 1.9 mu M. The construction and expression of recombinant P450 4A1 containing a six-member polyhistidine domain at the carboxy-terminus of the protein is described, Reconstitution experiments with this purified recombinant P450 4A1, DLPC, Chaps, b(5), and purified NADPH-P450 reductase show results similar to those obtained with the purified fusion protein, albeit at lower turnover rates. The requirement for normal-phase HPLC in resolving the metabolites formed during lauric acid metabolism is demonstrated. (C) 1996 Academic Press, Inc.