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非対称な触媒に対する頑丈でエナチオセレクティブなベイエル・ヴィリゲル単酸化酵素の実験室での進化
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
まとめ
研究者らは,熱安定性フェニラセトンモノオキシゲナーゼ (PAMO) 酵素を設計して,その基板の範囲を広げました. 新しい変異体は,ケトン解像度に挑戦するために高い活性とエナチオセレクティブ性を発揮し,親酵素の安定性を保持します.
科学分野:
- バイオカタリスと酵素工学
- 有機化学 オーガニック・ケミストリー
- プロテイン工学は,タンパク質の
背景:
- フェニラセトンモノオキシゲネーゼ (PAMO) は熱安定性酵素で,エナチオセレクティブのベイヤー-ヴィリガー反応に最適です.
- 野生型のPAMOは限られた基板範囲を示し,主にフェニラセトンおよび類似のアナログを受け入れます.
- PAMOの基板範囲とエナチオ選択性を拡大するための以前のタンパク質エンジニアリングの努力は,限られた成功を収めました.
研究 の 目的:
- より広い範囲のケトン基板に対する強化された活性とエナチオ選択性を持つ新しいPAMO変異体を開発する.
- 酵素機能を改善するために特定のタンパク質領域をターゲットにした新しい実験室進化戦略を調査する.
- 野生型のPAMOの固有の熱安定性をエンジニアリングされた変種で維持するために.
主な方法:
- 八つのベイエル・ヴィリゲル単酸化酵素の配列調整を用いたバイオインフォマティクスアプローチを採用しました.
- PAMOの統合X線構造データと,潜在的なランダム化部位を特定するための誘導フィットドッキング.
- 酵素の特定のループ領域に保存されたプロリン残基を標的とした集中型ライブラリ生成.
主要な成果:
- 開発されたPAMO変異体は,様々な2-アリルおよび2-アルキルサイクロヘクサノンを分解する上で,著しく高い活性とエナチオセレクティブ性を示しています.
- エンジニアリングによるミュータントは,構造的に異なるバイサイクルケトンを成功裏に処理し,野生型のPAMOを超えて基板の受容性を拡大しました.
- 最も効果的な変異体は,親野生型PAMOの特異的な熱安定性を保持しました.
結論:
- 保存されたプロリン部位をターゲットにした新しい実験室進化戦略は,PAMOの触媒性能を高めるのに非常に効果的です.
- エンジニアリングされたPAMOの変種は,多様なケトン基板のエナチオセレクティブバイエル-ヴィリガー酸化のための有望なバイオカタリストを提供します.
- "プロリン仮説"は,この種の酵素に関する将来の酵素進化研究において,さらなる調査を正当化している.
関連する概念動画
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...