传统的Kemp消除反应可以通过氢氧化钾和三烷基胺等碱性物质,催化底物苯并异恶唑开环生成产物2-氰基苯酚.三十年来,Kemp消除反应一直被用作模式反应来设计或定向进化新型生物酶催化剂,从而揭示未知的酶催化机制的复杂性,增强对酶催化机...传统的Kemp消除反应可以通过氢氧化钾和三烷基胺等碱性物质,催化底物苯并异恶唑开环生成产物2-氰基苯酚.三十年来,Kemp消除反应一直被用作模式反应来设计或定向进化新型生物酶催化剂,从而揭示未知的酶催化机制的复杂性,增强对酶催化机制的理解.目前科研人员使用不同的蛋白作为骨架设计能够高效催化Kemp消除反应的人工酶.例如Hilvert及Mayo等基于人工酶HG3.17,设计获得了Kemp消除酶,可以催化5-硝基苯并异恶唑生成产物2-氰基-4-硝基苯酚.通过17轮定向进化获得的最终突变体展现出与天然酶相近的催化活性(k_(cat)/Km=230000 L mol^(-1)s^(-1);k_(cat)=700 s^(-1)).该研究不仅表明蛋白质工程可以进化出高效的生物酶,量子力学/分子力学(QM/MM)分析还揭示了突变体催化活性提高的分子机制.与酸碱催化的Kemp消除反应不同,最近Korendovych等报道以肌红蛋白作为骨架基于氧化还原机制的Kemp消除反应,通过开发一种独特的基于核磁共振(NMR)的蛋白质定向进化技术,快速鉴定出热点氨基酸位点并获得了高催化活性的人工酶突变体,其同样达到了天然酶的催化活性.此前我们研究(Nat.Commun.,2017,8,14876)发现,与传统的酸碱催化机制完全不同,细胞色素P450-BM3能够通过氧化还原的机制催化Kemp消除反应.本文继续以P450-BM3为蛋白骨架,对其进一步改进以提高催化活性.以P450-BM3突变体F87G(kcat=3.0s^(-1))为模板,借助双密码子(酪氨酸和赖氨酸,Y-K)饱和突变策略,对其活性中心的六个关键氨基酸位点进行组合突变,经过筛选获得了活性大幅度提高的三突变体F87G/L75Y/T438K(k_(cat)=27.4s^(-1)).为进一步解析其催化活性提高的分子机制,首先对该突变体与底物复合物的晶体结构进行了解析,结果发现底物在突变体活性口袋的构象与野生型P450-BM3完全不同,底物的硝基指向了Heme辅基.对其进行了QM/MM计算研究,发现Heme-Fe(II)首先将电子转移到底物并与硝基配位,随后,有效促进了底物N-O键还原裂解,并使生成的中间产物Heme-Fe(III)-NO_(2)和苯氧基阴离子更稳定,最后,通过键的旋转和质子转移生成产物2-氰基-4-硝基苯酚.由此可见,同一P450酶的不同突变体能够以两种不同的底物结合模式以及氧化还原机制来催化Kemp消除反应.综上,本文获得了P450酶催化Kemp消除反应构效关系和催化机制新的认识,为进一步改造该酶提高其催化活性提供借鉴.展开更多
Intensive past research has demonstrated that cytochrome P450 monooxygenases are involved in the biosynthesis of numerous natural products and in human metabolism of various pharmaceuticals [1]. This inspired syntheti...Intensive past research has demonstrated that cytochrome P450 monooxygenases are involved in the biosynthesis of numerous natural products and in human metabolism of various pharmaceuticals [1]. This inspired synthetic organic and pharmaceutical chemists to test them as catalysts in the oxidative hydroxylation of steroids. Unfortunately, wildtype P450s generally proved to show low levels of regio-and diastereoselectivity as well as suboptimal activity.展开更多
The capture and transformation of the greenhouse gas CO_(2) into high-value products is currently of paramount importance worldwide,with many different approaches being conceived and tested[1-6].The transformation CO_...The capture and transformation of the greenhouse gas CO_(2) into high-value products is currently of paramount importance worldwide,with many different approaches being conceived and tested[1-6].The transformation CO_(2) into carbohydrates is a particularly intriguing idea,but putting it into reality is challenging due to the necessary complete control in the creation of stereo-centers and ensuring sufficient yields.Thus far,the results are not overly convincing[7].The inspiration for solving this fundamental problem is manifold,inter alia,because carbohydrates(sugars)contribute significantly to energy storage and provide carriers for human living,especially important biologically active substances.They also constitute the main resource for industrial biomanufacturing.展开更多
This book[1]begins with an introductory chapter in which the history of directed enzyme evolution is briefly presented and the different gene mutagenesis techniques are outlined,followed by a comprehensive chapter des...This book[1]begins with an introductory chapter in which the history of directed enzyme evolution is briefly presented and the different gene mutagenesis techniques are outlined,followed by a comprehensive chapter describing medium-and high-throughput screening systems for assaying stereoselectivity and activity.展开更多
文摘传统的Kemp消除反应可以通过氢氧化钾和三烷基胺等碱性物质,催化底物苯并异恶唑开环生成产物2-氰基苯酚.三十年来,Kemp消除反应一直被用作模式反应来设计或定向进化新型生物酶催化剂,从而揭示未知的酶催化机制的复杂性,增强对酶催化机制的理解.目前科研人员使用不同的蛋白作为骨架设计能够高效催化Kemp消除反应的人工酶.例如Hilvert及Mayo等基于人工酶HG3.17,设计获得了Kemp消除酶,可以催化5-硝基苯并异恶唑生成产物2-氰基-4-硝基苯酚.通过17轮定向进化获得的最终突变体展现出与天然酶相近的催化活性(k_(cat)/Km=230000 L mol^(-1)s^(-1);k_(cat)=700 s^(-1)).该研究不仅表明蛋白质工程可以进化出高效的生物酶,量子力学/分子力学(QM/MM)分析还揭示了突变体催化活性提高的分子机制.与酸碱催化的Kemp消除反应不同,最近Korendovych等报道以肌红蛋白作为骨架基于氧化还原机制的Kemp消除反应,通过开发一种独特的基于核磁共振(NMR)的蛋白质定向进化技术,快速鉴定出热点氨基酸位点并获得了高催化活性的人工酶突变体,其同样达到了天然酶的催化活性.此前我们研究(Nat.Commun.,2017,8,14876)发现,与传统的酸碱催化机制完全不同,细胞色素P450-BM3能够通过氧化还原的机制催化Kemp消除反应.本文继续以P450-BM3为蛋白骨架,对其进一步改进以提高催化活性.以P450-BM3突变体F87G(kcat=3.0s^(-1))为模板,借助双密码子(酪氨酸和赖氨酸,Y-K)饱和突变策略,对其活性中心的六个关键氨基酸位点进行组合突变,经过筛选获得了活性大幅度提高的三突变体F87G/L75Y/T438K(k_(cat)=27.4s^(-1)).为进一步解析其催化活性提高的分子机制,首先对该突变体与底物复合物的晶体结构进行了解析,结果发现底物在突变体活性口袋的构象与野生型P450-BM3完全不同,底物的硝基指向了Heme辅基.对其进行了QM/MM计算研究,发现Heme-Fe(II)首先将电子转移到底物并与硝基配位,随后,有效促进了底物N-O键还原裂解,并使生成的中间产物Heme-Fe(III)-NO_(2)和苯氧基阴离子更稳定,最后,通过键的旋转和质子转移生成产物2-氰基-4-硝基苯酚.由此可见,同一P450酶的不同突变体能够以两种不同的底物结合模式以及氧化还原机制来催化Kemp消除反应.综上,本文获得了P450酶催化Kemp消除反应构效关系和催化机制新的认识,为进一步改造该酶提高其催化活性提供借鉴.
文摘Intensive past research has demonstrated that cytochrome P450 monooxygenases are involved in the biosynthesis of numerous natural products and in human metabolism of various pharmaceuticals [1]. This inspired synthetic organic and pharmaceutical chemists to test them as catalysts in the oxidative hydroxylation of steroids. Unfortunately, wildtype P450s generally proved to show low levels of regio-and diastereoselectivity as well as suboptimal activity.
文摘The capture and transformation of the greenhouse gas CO_(2) into high-value products is currently of paramount importance worldwide,with many different approaches being conceived and tested[1-6].The transformation CO_(2) into carbohydrates is a particularly intriguing idea,but putting it into reality is challenging due to the necessary complete control in the creation of stereo-centers and ensuring sufficient yields.Thus far,the results are not overly convincing[7].The inspiration for solving this fundamental problem is manifold,inter alia,because carbohydrates(sugars)contribute significantly to energy storage and provide carriers for human living,especially important biologically active substances.They also constitute the main resource for industrial biomanufacturing.
文摘This book[1]begins with an introductory chapter in which the history of directed enzyme evolution is briefly presented and the different gene mutagenesis techniques are outlined,followed by a comprehensive chapter describing medium-and high-throughput screening systems for assaying stereoselectivity and activity.
