• 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Dalton transactions: An international journal of inorganic chemistry >Engineering cytochrome P450cam into an alkane hydroxylase
【24h】

Engineering cytochrome P450cam into an alkane hydroxylase

机译：将细胞色素P450cam工程化为烷烃羟化酶

获取原文
获取原文并翻译 | 示例
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

The haem monooxygenase cytochrome P450cam from Pseudomonas putida has been engineered into an alkane hydroxylase. Active site amino acid residues were substitued with residues that have bulkier and more hydrophobic side-chains. The residues F87, Y96, V247 and V396, which are further away from the haem, were targeted first for substitution in order to constrain the small alkanes n-btane and propane to bind closer to the haem. We found that just two mutations could increase the alkane oxidation activity of P450cam by two orders of magnitude. The F87W/Y96F/V247L triple mutant was then used as a basis for introducing further substitutions, at the residues T101, L244, V395 and D297 which are closer to the haem, to improve the enzyme/alkane fit and hence the alkane hydroxylase activity. The F87W/Y96F/T101L/V247L mutant oxidised n-butane with a catalytic turnover rate of 755 nmol (nmol P450 cam)~(-1) (min)~(-1), which is comparable to the camphor oxidation activity of the wild-type (1000 min~(-1)). The F87W/Y96F/T101L/L244M/V247L mutant had lower n-butane oxidation activity but the highest propane oxidation rate (176 min~(-1)) of the P450cam enzymes studied. All P450 cam enzymes gave 2-butanol and 2-propanol as the only products. Determination of the extent of uncoupling showed that hydrogen peroxide generation was the dominant uncoupling mechanism. The data indicate that further mutations at residues higher up in the active site are required to localise the substrates close to the haem and to reduce substrate mobility. These next-generation mutants will have higher activity, and may be able to catalyse the oxidation of ethane and methane.
机译：来自恶臭假单胞菌的血红素单加氧酶细胞色素P450cam已被工程化为烷烃羟化酶。活性位点氨基酸残基被具有更大和更疏水的侧链的残基取代。远离血红素的残基F87，Y96，V247和V396首先被靶向取代，以限制小烷烃正丁烷和丙烷更紧密地结合到血红素上。我们发现只有两个突变可以将P450cam的烷烃氧化活性提高两个数量级。然后将F87W / Y96F / V247L三重突变体用作在更靠近血红素的残基T101，L244，V395和D297处引入进一步取代的基础，以提高酶/烷烃适合度，从而改善烷烃羟化酶活性。 F87W / Y96F / T101L / V247L突变型氧化正丁烷的催化转化率为755 nmol（nmol P450 cam）〜（-1）（min）〜（-1），可与苯酚的樟脑氧化活性相媲美。野生型（1000 min〜（-1））。在研究的P450cam酶中，F87W / Y96F / T101L / L244M / V247L突变体具有较低的正丁烷氧化活性，但丙烷的氧化速率最高（176 min〜（-1））。所有P450凸轮酶都仅产生2-丁醇和2-丙醇。解偶联程度的测定表明过氧化氢的产生是主要的解偶联机理。数据表明，需要在活性位点较高的残基处进行进一步的突变，以将底物定位在血红素附近并降低底物的迁移率。这些下一代突变体将具有更高的活性，并且可能能够催化乙烷和甲烷的氧化。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号