7exs

Thermomicrobium roseum sarcosine oxidase mutant - S320R

spinqualitylabelsall models 7exs, resolution 1.42Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 7exs is a 1 chain structure with sequence from Thermomicrobium roseum DSM 5159. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 1.42Å Ligands: CL, FAD, GOL, PO4 Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Function B9L163_THERP Publication Abstract from PubMed 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 degrees 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 non-conserved residues of key microdomains-including the catalytic loop, coenzyme pocket, substrate pocket, and entrance site-were 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. Recreating the natural evolutionary trend in key microdomains provides an effective strategy for engineering of a thermomicrobial N-demethylase.,Xin Y, Shen C, Tang M, Guo Z, Shi Y, Gu Z, Shao J, Zhang L J Biol Chem. 2022 Feb 3:101656. doi: 10.1016/j.jbc.2022.101656. PMID:35124004 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See Also Sarcosine oxidase

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