9GKT

Crystal structure of artificial enzyme LmrR_pAF variant RGN

9GKT の概要

• エントリーDOI: 10.2210/pdb9gkt/pdb
• 分子名称: Transcriptional regulator, PadR-like family (2 entities in total)
• 機能のキーワード: artificial enzyme, unnatural amino acid, 4-aminophenylalanine, directed evolution, dna binding protein
• 由来する生物種: Lactococcus cremoris subsp. cremoris MG1363
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 29501.24

構造登録者

Thunnissen, A.M.W.H.,Leveson-Gower, R.B.,Rozeboom, H.J.,Roelfes, G. (登録日: 2024-08-26, 公開日: 2025-02-26)

主引用文献

Leveson-Gower, R.B.,Tiessler-Sala, L.,Rozeboom, H.J.,Thunnissen, A.W.H.,Marechal, J.D.,Roelfes, G.
Evolutionary Specialization of a Promiscuous Designer Enzyme.
Acs Catalysis, 15:1544-1552, 2025

PubMed Abstract: The evolution of a promiscuous enzyme for its various activities often results in catalytically specialized variants. This is an important natural mechanism to ensure the proper functioning of natural metabolic networks. It also acts as both a curse and blessing for enzyme engineers, where enzymes that have undergone directed evolution may exhibit exquisite selectivity at the expense of a diminished overall catalytic repertoire. We previously performed two independent directed evolution campaigns on a promiscuous designer enzyme that leverages the unique properties of a noncanonical amino acid (ncAA) -aminophenylalanine (pAF) as catalytic residue, resulting in two evolved variants which are both catalytically specialized. Here, we combine mutagenesis, crystallography, and computation to reveal the molecular basis of the specialization phenomenon. In one evolved variant, an unexpected change in quaternary structure biases substrate dynamics to promote enantioselective catalysis, while the other demonstrates synergistic cooperation between natural side chains and the pAF residue to form semisynthetic catalytic machinery.

PubMed: 39944761
DOI: 10.1021/acscatal.4c06409

実験手法

X-RAY DIFFRACTION (2.45 Å)