实验室进化强大的和enantioselective贝耶-维利格单氧基酶对不对称的催化剂
1Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mulheim an der Ruhr, Germany. reetz@mpi-muelheim.mpg.de
Journal of the American Chemical Society
|October 8, 2009
概括
研究人员设计了热稳定的甲单氧化酶 (PAMO) 酶,以扩大其基质范围. 新突变对挑战子分辨率表现出高活性和酶选择性,保持母酶稳定性.
科学领域:
- 生物催化和酶工程 生物催化和酶工程
- 有机化学 有机化学
- 蛋白质工程是指蛋白质工程.
背景情况:
- 甲单氧酶 (PAMO) 是一种热稳定酶,非常适合对抗选择性贝耶-维利格反应.
- 野生类型的PAMO表现出有限的基质范围,主要接受基和类似的类似物.
- 之前的蛋白质工程努力扩大PAMO的基质范围和酶选择性取得了有限的成功.
研究的目的:
- 为更广泛的基质开发具有增强活性和酶选择性的新型PAMO突变.
- 研究一种新的实验室进化策略,针对特定的蛋白质区域,以改善酶功能.
- 为了保持野生类型PAMO在工程变体中固有的热稳定性.
主要方法:
- 采用生物信息学方法,使用八种Baeyer-Villiger单氧酶的序列对齐.
- 整合了PAMO的X射线结构数据和诱导的合适对接,以确定潜在的随机化地点.
- 聚焦图书馆生成,针对酶的特定循环区域中的保存的林残留物.
主要成果:
- 开发了PAMO突变,显示出显著更高的活性和对各种2-和2-环素的解脱而产生反选择性.
- 工程突变物成功地处理了结构上不同的双循环,扩大了基质接受度超出了野生类型的PAMO.
- 最有效的突变保留了原始野生类型PAMO的特殊热稳定性特征.
结论:
- 新的实验室进化策略针对保留的普罗林位点,对提高PAMO的催化性能非常有效.
- 工程 PAMO 变体为各种基质的 enantioselective Baeyer-Villiger 氧化提供了有希望的生物催化剂.
- 这种"proline假设"需要在未来对这一类酶的酶进化研究中进行进一步的研究.
相关概念视频
01:22Reactions of Aldehydes and Ketones: Baeyer–Villiger Oxidation
Baeyer–Villiger oxidation converts aldehydes to carboxylic acids and ketones to esters. The reaction uses peroxy acids or peracids and is often catalyzed by acid. The reaction is named after its pioneers, Adolf von Baeyer and Victor Villiger. The reaction is achieved by a wide range of peracids such as m-chloroperoxybenzoic acid (mCPBA), perbenzoic acid (C6H5COOOH), peracetic acid (CH3COOOH), hydrogen peroxide (H2O2), and tert-butyl hydroperoxide (t-BuOOH).
The carbonyl center is activated by...
The carbonyl center is activated by...
02:36Regioselectivity and Stereochemistry of Hydroboration
A significant aspect of hydroboration–oxidation is the regio- and stereochemical outcome of the reaction.
Hydroboration proceeds in a concerted fashion with the attack of borane on the π bond, giving a cyclic four-centered transition state. The –BH2 group is bonded to the less substituted carbon and –H to the more substituted carbon. The concerted nature requires the simultaneous addition of –H and –BH2 across the same face of the alkene giving syn stereochemistry.
Hydroboration proceeds in a concerted fashion with the attack of borane on the π bond, giving a cyclic four-centered transition state. The –BH2 group is bonded to the less substituted carbon and –H to the more substituted carbon. The concerted nature requires the simultaneous addition of –H and –BH2 across the same face of the alkene giving syn stereochemistry.
02:17Reduction of Alkenes: Asymmetric Catalytic Hydrogenation
Catalytic hydrogenation of alkenes is a transition-metal catalyzed reduction of the double bond using molecular hydrogen to give alkanes. The mode of hydrogen addition follows syn stereochemistry.
The metal catalyst used can be either heterogeneous or homogeneous. When hydrogenation of an alkene generates a chiral center, a pair of enantiomeric products is expected to form. However, an enantiomeric excess of one of the products can be facilitated using an enantioselective reaction or an...
The metal catalyst used can be either heterogeneous or homogeneous. When hydrogenation of an alkene generates a chiral center, a pair of enantiomeric products is expected to form. However, an enantiomeric excess of one of the products can be facilitated using an enantioselective reaction or an...
01:33Regioselective Formation of Enolates
As depicted in the figure below, the unsymmetrical ketones can form two possible enolates: less substituted or more substituted enolates. Usually, the thermodynamic enolates are formed from the more substituted α-carbon atom, while the kinetic enolates are formed faster by deprotonation from the less substituted position. The thermodynamic enolates have lower energy, so they are more stable. But the energy required to form kinetic enolates is less.
03:08Hydroboration-Oxidation of Alkenes
In addition to the oxymercuration–demercuration method, which converts the alkenes to alcohols with Markovnikov orientation, a complementary hydroboration-oxidation method yields the anti-Markovnikov product. The hydroboration reaction, discovered in 1959 by H.C. Brown, involves the addition of a B–H bond of borane to an alkene giving an organoborane intermediate. The oxidation of this intermediate with basic hydrogen peroxide forms an alcohol.
01:24Diels–Alder Reaction: Characteristics of Dienophiles
In a Diels–Alder reaction, the diene is usually an electron-rich system and acts as a nucleophile, whereas the dienophile is electron-deficient and functions as an electrophile. Much like the diene, the nature of the dienophile significantly impacts the outcome of the reaction.
Characteristics of Dienophiles
Generally, the best dienophiles are alkenes containing electron-withdrawing substituents such as carbonyl, nitrile, and nitro groups. The feasibility of a Diels–Alder reaction depends on...
Characteristics of Dienophiles
Generally, the best dienophiles are alkenes containing electron-withdrawing substituents such as carbonyl, nitrile, and nitro groups. The feasibility of a Diels–Alder reaction depends on...