4O5T

Crystal structure of Diels-Alderase CE20 in complex with a product analog

4O5T の概要

• エントリーDOI: 10.2210/pdb4o5t/pdb
• 関連するPDBエントリー: 1E1A 3I1C 3U0S 4O5S
• 分子名称: Diisopropyl-fluorophosphatase, 4-{[2-(phosphonooxy)ethyl]carbamoyl}benzyl [(1R,6S)-6-(dimethylcarbamoyl)cyclohex-2-en-1-yl]carbamate (3 entities in total)
• 機能のキーワード: protein engineering, computer-aided design, diels-alder reaction, enzyme design, directed evolution, substrate specificity, beta-propeller, helix-loop-helix, de novo protein, artificial catalyst, diels-alderase, catalyst for cycloaddition, hydrolase, enzyme-product analog complex'
• 由来する生物種: Loligo vulgaris (Common European squid)
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 75539.05

構造登録者

Beck, T.,Preiswerk, N.,Mayer, C.,Hilvert, D. (登録日: 2013-12-20, 公開日: 2014-06-04, 最終更新日: 2023-11-08)

主引用文献

Preiswerk, N.,Beck, T.,Schulz, J.D.,Milovnik, P.,Mayer, C.,Siegel, J.B.,Baker, D.,Hilvert, D.
Impact of scaffold rigidity on the design and evolution of an artificial Diels-Alderase.
Proc.Natl.Acad.Sci.USA, 111:8013-8018, 2014

PubMed Abstract: By combining targeted mutagenesis, computational refinement, and directed evolution, a modestly active, computationally designed Diels-Alderase was converted into the most proficient biocatalyst for [4+2] cycloadditions known. The high stereoselectivity and minimal product inhibition of the evolved enzyme enabled preparative scale synthesis of a single product diastereomer. X-ray crystallography of the enzyme-product complex shows that the molecular changes introduced over the course of optimization, including addition of a lid structure, gradually reshaped the pocket for more effective substrate preorganization and transition state stabilization. The good overall agreement between the experimental structure and the original design model with respect to the orientations of both the bound product and the catalytic side chains contrasts with other computationally designed enzymes. Because design accuracy appears to correlate with scaffold rigidity, improved control over backbone conformation will likely be the key to future efforts to design more efficient enzymes for diverse chemical reactions.

PubMed: 24847076
DOI: 10.1073/pnas.1401073111

実験手法

X-RAY DIFFRACTION (2.9 Å)