Shet M S, Fisher C W, Estabrook R W
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235-9038, USA.
Arch Biochem Biophys. 1997 Mar 1;339(1):218-25. doi: 10.1006/abbi.1996.9868.
Studies are reported showing that recombinant P450c17, coexpressed with rat NADPH-P450 reductase or expressed as a fusion protein containing the domain of the P450 linked to the domain of NADPH-P450 reductase, function effectively in intact Escherichia coli cells. Progesterone is rapidly hydroxylated by transformed E. coli cells at rates as rapid as 50 nmol of steroid hydroxylated/min/nmol of P450 at 37 degrees C. This rate measured in vivo equals or exceeds the best rates we have measured when reconstituting progesterone hydroxylase activity in vitro using purified recombinant bovine P450c17 and purified recombinant rat NADPH-P450 reductase. The limits imposed in vivo by the availability of reducing equivalents (NADPH) and molecular oxygen are identified by showing the nearly fivefold increase in hydroxylation activity when glucose is present and the tendency for the constitutive respiratory activity of E. coli to limit the availability of oxygen required for the P450-catalyzed reaction. The rate of progesterone metabolism is about 200 times faster by P450c17 coexpressed with NADPH-P450 reductase than when P450c17 functions with the constitutive electron transfer system of E. coli (flavodoxin and flavodoxin reductase). Expression of the fusion protein, termed rF450[mBov17A/mRatOR]L1, results in a rate of progesterone metabolism in vivo at 37 degrees C of about 15 nmol of steroid hydroxylated/min/nmol of P450. Pregnenolone is actively metabolized to dehydroepiandrosterone at rates similar to those seen when the P450 activity is reconstituted in vitro with cytochrome b5. Experiments are described showing that the limited solubility of progesterone in water imposes a limit on the extent of steroid hydroxylated. The practicality of this type of P450-containing system for the bioconversion of large amounts of a chemical for the manufacture of speciality chemicals is discussed.
有研究报告显示，重组P450c17与大鼠NADPH - P450还原酶共表达，或以包含与NADPH - P450还原酶结构域相连的P450结构域的融合蛋白形式表达时，能在完整的大肠杆菌细胞中有效发挥作用。在37℃时，经转化的大肠杆菌细胞能迅速将孕酮羟基化，速率可达50 nmol类固醇羟基化/分钟/nmol P450。在体内测得的该速率等于或超过我们使用纯化的重组牛P450c17和纯化的重组大鼠NADPH - P450还原酶在体外重建孕酮羟化酶活性时测得的最佳速率。通过显示当存在葡萄糖时羟化活性几乎增加五倍以及大肠杆菌组成型呼吸活性限制P450催化反应所需氧气可用性的趋势，确定了体内还原当量（NADPH）和分子氧可用性所施加的限制。与NADPH - P450还原酶共表达的P450c17对孕酮的代谢速率比P450c17与大肠杆菌组成型电子传递系统（黄素氧还蛋白和黄素氧还蛋白还原酶）一起发挥作用时快约200倍。称为rF450[mBov17A/mRatOR]L1的融合蛋白的表达导致在37℃时体内孕酮代谢速率约为15 nmol类固醇羟基化/分钟/nmol P450。孕烯醇酮以与用细胞色素b5在体外重建P450活性时相似的速率被积极代谢为脱氢表雄酮。所描述的实验表明，孕酮在水中的有限溶解度对类固醇羟基化的程度施加了限制。讨论了这种含P450系统用于大量化学物质生物转化以制造特种化学品的实用性。
