Engineering of cytochrome P450 3A4 for enhanced peroxide-mediated substrate oxidation using directed evolution and site-directed mutagenesis
文献类型：期刊论文
| 作者 | Kumar, Santosh; Liu, Hong ; Halpert, James R.
|
| 刊名 | DRUG METABOLISM AND DISPOSITION
 |
| 出版日期 | 2006-12 |
| 卷号 | 34期号:12页码:1958-1965 |
| ISSN号 | 0090-9556 |
| DOI | 10.1124/dmd.106.012054 |
| 文献子类 | Article; Proceedings Paper |
| 英文摘要 | CYP3A4 has been subjected to random and site-directed mutagenesis to enhance peroxide-supported metabolism of several substrates. Initially, a high-throughput screening method using whole cell suspensions was developed for H2O2-supported oxidation of 7-benzyloxyquinoline. Random mutagenesis by error-prone polymerase chain reaction and activity screening yielded several CYP3A4 mutants with enhanced activity. L216W and F228I showed a 3-fold decrease in K-m, (HOOH) and a 2.5-fold increase in k(cat)/K-m, (HOOH) compared with CYP3A4. Subsequently, T309V and T309A were created based on the observation that T309V in CYP2D6 has enhanced cumene hydroperoxide (CuOOH)-supported activity. T309V and T309A showed a > 6- and 5-fold higher kcat/Km, CuOOH than CYP3A4, respectively. Interestingly, L216W and F228I also exhibited, respectively, a > 4- and a > 3- fold higher k(cat)/K-m, (CuOOH) than CYP3A4. Therefore, several multiple mutants were constructed from rationally designed and randomly isolated mutants; among them, F228I/T309A showed an 11-fold higher k(cat)/K-m, (CuOOH) than CYP3A4. Addition of cytochrome b(5), which is known to stimulate peroxide-supported activity, enhanced the k(cat)/K-m, (CuOOH) of CYP3A4 by 4- to 7-fold. When the mutants were tested with other substrates, T309V and T433S showed enhanced k(cat)/K-m, (CuOOH) with 7-benzyloxy-4-(trifluoromethyl)coumarin and testosterone, respectively, compared with CYP3A4. In addition, in the presence of cytochrome b5, T433S has the potential to produce milligram quantities of 6 beta-hydroxytestosterone through peroxide-supported oxidation. In conclusion, a combination of random and site-directed mutagenesis approaches yielded CYP3A4 enzymes with enhanced peroxide-supported metabolism of several substrates. |
| WOS关键词 | CYP3A4 ACTIVE-SITE ; SCANNING MUTAGENESIS ; CRYSTAL-STRUCTURE ; RECOGNITION SITE ; DRUG-METABOLISM ; B(5) ; REDUCTASE ; MUTANT ; 2B4 ; COOPERATIVITY |
| 资助项目 | NIEHS NIH HHS[ES06676] ; NIGMS NIH HHS[GM54995] ; NIGMS NIH HHS[R37 GM054995] |
| WOS研究方向 | Pharmacology & Pharmacy |
| 语种 | 英语 |
| WOS记录号 | WOS:000242374700004 |
| 出版者 | AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS |
| 源URL | [http://119.78.100.183/handle/2S10ELR8/273432]  |
| 专题 | 中国科学院上海药物研究所 |
| 通讯作者 | Kumar, Santosh |
| 作者单位 | 1.Univ Texas, Med Branch, Dept Pharmacol & Toxicol, Galveston, TX 77555 USA 2.Chinese Acad Sci, Shanghai Inst Mat Med, Ctr Drug Discovery & Design, Beijing 100864, Peoples R China |
| 推荐引用方式 GB/T 7714 | Kumar, Santosh,Liu, Hong,Halpert, James R.. Engineering of cytochrome P450 3A4 for enhanced peroxide-mediated substrate oxidation using directed evolution and site-directed mutagenesis[J]. DRUG METABOLISM AND DISPOSITION,2006,34(12):1958-1965. |
| APA | Kumar, Santosh,Liu, Hong,&Halpert, James R..(2006).Engineering of cytochrome P450 3A4 for enhanced peroxide-mediated substrate oxidation using directed evolution and site-directed mutagenesis.DRUG METABOLISM AND DISPOSITION,34(12),1958-1965. |
| MLA | Kumar, Santosh,et al."Engineering of cytochrome P450 3A4 for enhanced peroxide-mediated substrate oxidation using directed evolution and site-directed mutagenesis".DRUG METABOLISM AND DISPOSITION 34.12(2006):1958-1965. |
入库方式： OAI收割
来源：上海药物研究所
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