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7exs

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== Publication Abstract from PubMed ==
== Publication Abstract from PubMed ==
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Thermomicrobium roseum sarcosine oxidase (TrSOX) was a N-demethylase with specific substrate chiral selectivity, outstanding thermostability and environmental resistance. To promote the expression of TrSOX in Bacillus subtilis W600, the HpaII promoter of pMA5 plasmid was replaced by constitutive or inducible promoters. Through orthogonal experiment, the expression process was optimized, B. subtilis W600 cells containing pMA5-Pxyl-trSOX plasmid were cultivated until OD600nm reached 2.0 and were then induced with 1.6% xylose at 37 degrees C for 2 h, and the native environment of T. roseum was simulated by heating at 80 degrees C, with the productivity of TrSOX increased from ~8.3 to ~66.7 mug/g wet cells; and the simulated high temperature was the key switch for the final folding. To reduce the surface hydrophobicity, a S320R mutant was built to form a hydrophilic lid around the entrance of the substrate pocket, and the yield of TrSOX (S320R) was ~163.0 mug/g wet cells, approximately 20 folds as that in the initial expression system. This mutant revealed the similar secondary structure, stability, resistance, chiral substrate selectivity and optimal reaction environment with wild type TrSOX; however, the N-demethylation activities for amino acid derivative substrates were dramatically increased, while those for hydrophobic non-amino acid compounds were repressed.
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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.
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Promote the expression and corrected folding of an extremely stable N-demethylase by promoter reconstruction, native environment simulation and surface design.,Gao Q, Shao J, Tang M, Xin Y, Zhang L Int J Biol Macromol. 2021 May 1;178:434-443. doi: 10.1016/j.ijbiomac.2021.02.176., Epub 2021 Feb 27. PMID:33647338<ref>PMID:33647338</ref>
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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<ref>PMID:35124004</ref>
From MEDLINE&reg;/PubMed&reg;, a database of the U.S. National Library of Medicine.<br>
From MEDLINE&reg;/PubMed&reg;, a database of the U.S. National Library of Medicine.<br>

Revision as of 08:37, 23 February 2022

Thermomicrobium roseum sarcosine oxidase mutant - S320R

PDB ID 7exs

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