64199-88-8Relevant articles and documents
Recreating the natural evolutionary trend in key microdomains provides an effective strategy for engineering of a thermomicrobial N-demethylase
Gu, Zhenghua,Guo, Zitao,Shao, Jun,Shen, Chen,Shi, Yi,Tang, Mengwei,Xin, Yu,Zhang, Liang
, (2022/03/09)
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.
Crystal structure of substrate-bound bifunctional proline racemase/hydroxyproline epimerase from a hyperthermophilic archaeon
Watanabe, Yasunori,Watanabe, Seiya,Itoh, Yoshika,Watanabe, Yasuo
, p. 135 - 140 (2019/02/19)
The hypothetical OCC_00372 protein from Thermococcus litoralis is a member of the ProR superfamily from hyperthermophilic archaea and exhibits unique bifunctional proline racemase/hydroxyproline 2-epimerase activity. However, the molecular mechanism of th
A unique cis -3-hydroxy- l -proline dehydratase in the enolase superfamily
Zhang, Xinshuai,Kumar, Ritesh,Vetting, Matthew W.,Zhao, Suwen,Jacobson, Matthew P.,Almo, Steven C.,Gerlt, John A.
supporting information, p. 1388 - 1391 (2015/03/05)
The genome of Labrenzia aggregata IAM 12614 encodes an uncharacterized member of the muconate lactonizing enzyme (MLE) subgroup of the enolase superfamily (UniProt ID A0NXQ8). The gene encoding A0NXQ8 is located between genes that encode members of the proline racemase superfamily, 4R-hydroxyproline 2-epimerase (UniProt ID A0NXQ7; 4HypE) and trans-3-hydroxy-l-proline dehydratase (UniProt ID A0NXQ9; t3LHypD). A0NXQ8 was screened with a library of proline analogues; two reactions were observed with cis-3-hydroxy-l-proline (c3LHyp), competing 2-epimerization to trans-3-hydroxy-d-proline (1,1-proton transfer) and dehydration to δ1-pyrroline-2-carboxylate (β-elimination; c3LHyp dehydratase), with eventual total dehydration. The genome context encoding A0NXQ8 both (1) confirms its novel c3LHyp dehydratase function and (2) provides evidence for metabolic pathways that allow L. aggregata to utilize several isomeric 3- and 4-hydroxyprolines as sole carbon sources.
