8BP1

Crystal structure of BHMeHis1.0, an engineered enzyme for the Morita-Baylis-Hillman reaction

8BP1 の概要

• エントリーDOI: 10.2210/pdb8bp1/pdb
• 分子名称: BHMeHis1.0, DI(HYDROXYETHYL)ETHER, ACETATE ION, ... (6 entities in total)
• 機能のキーワード: morita-baylis-hillman, non-canonical amino acid, methylhistidine, de novo enzyme, biosynthetic protein
• 由来する生物種: synthetic construct
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 28005.64

構造登録者

Hardy, F.J. (登録日: 2022-11-15, 公開日: 2024-02-28, 最終更新日: 2026-03-04)

主引用文献

Hutton, A.E.,Foster, J.,Crawshaw, R.,Hardy, F.J.,Johannissen, L.O.,Lister, T.M.,Gerard, E.F.,Birch-Price, Z.,Obexer, R.,Hay, S.,Green, A.P.
A non-canonical nucleophile unlocks a new mechanistic pathway in a designed enzyme.
Nat Commun, 15:1956-1956, 2024

PubMed Abstract: Directed evolution of computationally designed enzymes has provided new insights into the emergence of sophisticated catalytic sites in proteins. In this regard, we have recently shown that a histidine nucleophile and a flexible arginine can work in synergy to accelerate the Morita-Baylis-Hillman (MBH) reaction with unrivalled efficiency. Here, we show that replacing the catalytic histidine with a non-canonical N-methylhistidine (MeHis23) nucleophile leads to a substantially altered evolutionary outcome in which the catalytic Arg124 has been abandoned. Instead, Glu26 has emerged, which mediates a rate-limiting proton transfer step to deliver an enzyme (BH1.8) that is more than an order of magnitude more active than our earlier MBHase. Interestingly, although MeHis23 to His substitution in BH1.8 reduces activity by 4-fold, the resulting His containing variant is still a potent MBH biocatalyst. However, analysis of the BH1.8 evolutionary trajectory reveals that the MeHis nucleophile was crucial in the early stages of engineering to unlock the new mechanistic pathway. This study demonstrates how even subtle perturbations to key catalytic elements of designed enzymes can lead to vastly different evolutionary outcomes, resulting in new mechanistic solutions to complex chemical transformations.

PubMed: 38438341
DOI: 10.1038/s41467-024-46123-z

実験手法

X-RAY DIFFRACTION (1.72 Å)