7EXS

Thermomicrobium roseum sarcosine oxidase mutant - S320R

7EXS の概要

• エントリーDOI: 10.2210/pdb7exs/pdb
• 分子名称: Sarcosine oxidase, FLAVIN-ADENINE DINUCLEOTIDE, GLYCEROL, ... (6 entities in total)
• 機能のキーワード: n-demethylase, extremely stable, chiral specificity, oxidoreductase
• 由来する生物種: Thermomicrobium roseum DSM 5159
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 43247.50

構造登録者

Xin, Y.,Shen, C.,Tang, M.W.,Shi, Y.,Guo, Z.T.,Gu, Z.H.,Shao, J.,Zhang, L. (登録日: 2021-05-28, 公開日: 2021-07-28, 最終更新日: 2023-11-29)

主引用文献

Xin, Y.,Shen, C.,Tang, M.,Guo, Z.,Shi, Y.,Gu, Z.,Shao, J.,Zhang, L.
Recreating the natural evolutionary trend in key microdomains provides an effective strategy for engineering of a thermomicrobial N-demethylase.
J.Biol.Chem., 298:101656-101656, 2022

PubMed Abstract: N-demethylases have been reported to remove the methyl groups on primary or secondary amines, which could further affect the properties and functions of biomacromolecules or chemical compounds; however, the substrate scope and the robustness of N-demethylases have not been systematically investigated. Here we report the recreation of natural evolution in key microdomains of the Thermomicrobium roseum sarcosine oxidase (TrSOX), an N-demethylase with marked stability (melting temperature over 100 °C) and enantioselectivity, for enhanced substrate scope and catalytic efficiency on -C-N- bonds. We obtained the structure of TrSOX by crystallization and X-ray diffraction (XRD) for the initial framework. The natural evolution in the nonconserved residues of key microdomains-including the catalytic loop, coenzyme pocket, substrate pocket, and entrance site-was then identified using ancestral sequence reconstruction (ASR), and the substitutions that accrued during natural evolution were recreated by site-directed mutagenesis. The single and double substitution variants catalyzed the N-demethylation of N-methyl-L-amino acids up to 1800- and 6000-fold faster than the wild type, respectively. Additionally, these single substitution variants catalyzed the terminal N-demethylation of non-amino-acid compounds and the oxidation of the main chain -C-N- bond to a -C=N- bond in the nitrogen-containing heterocycle. Notably, these variants retained the enantioselectivity and stability of the initial framework. We conclude that the variants of TrSOX are of great potential use in N-methyl enantiomer resolution, main-chain Schiff base synthesis, and alkaloid modification or degradation.

PubMed: 35124004
DOI: 10.1016/j.jbc.2022.101656

実験手法

X-RAY DIFFRACTION (1.42 Å)