8EUN

MicroED structure of an Aeropyrum pernix protoglobin metallo-carbene complex

8EUN の概要

• エントリーDOI: 10.2210/pdb8eun/pdb
• 関連するPDBエントリー: 8EUM
• EMDBエントリー: 28615 28616
• 分子名称: Protogloblin ApPgb, benzyl[3,3'-(7,12-diethenyl-3,8,13,17-tetramethylporphyrin-2,18-diyl-kappa~4~N~21~,N~22~,N~23~,N~24~)di(propanoato)(2-)]iron (3 entities in total)
• 機能のキーワード: microed, directed evolution, metal binding protein
• 由来する生物種: Aeropyrum pernix
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 46853.01

構造登録者

Danelius, E.,Gonen, T.,Unge, J.T. (登録日: 2022-10-19, 公開日: 2023-04-05, 最終更新日: 2024-05-22)

主引用文献

Danelius, E.,Porter, N.J.,Unge, J.,Arnold, F.H.,Gonen, T.
MicroED Structure of a Protoglobin Reactive Carbene Intermediate.
J.Am.Chem.Soc., 145:7159-7165, 2023

PubMed Abstract: Microcrystal electron diffraction (MicroED) is an emerging technique that has shown great potential for describing new chemical and biological molecular structures. Several important structures of small molecules, natural products, and peptides have been determined using methods. However, only a couple of novel protein structures have thus far been derived by MicroED. Taking advantage of recent technological advances, including higher acceleration voltage and using a low-noise detector in counting mode, we have determined the first structure of an protoglobin (Pgb) variant by MicroED using an AlphaFold2 model for phasing. The structure revealed that mutations introduced during directed evolution enhance carbene transfer activity by reorienting an α helix of Pgb into a dynamic loop, making the catalytic active site more readily accessible. After exposing the tiny crystals to the substrate, we also trapped the reactive iron-carbenoid intermediate involved in this engineered Pgb's new-to-nature activity, a challenging carbene transfer from a diazirine via a putative metallo-carbene. The bound structure discloses how an enlarged active site pocket stabilizes the carbene bound to the heme iron and, presumably, the transition state for the formation of this key intermediate. This work demonstrates that improved MicroED technology and the advancement in protein structure prediction now enable investigation of structures that was previously beyond reach.

PubMed: 36948184
DOI: 10.1021/jacs.2c12004

実験手法

ELECTRON CRYSTALLOGRAPHY (2.5 Å)