Recreating the natural evolutionary trend in key microdomains provides an effective strategy for engineering of a thermomicrobial N-demethylase.
Recreating the natural evolutionary trend in key microdomains provides an effective strategy for engineering of a thermomicrobial N-demethylase.
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DOI：
10.1016/j.jbc.2022.101656
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发表时间：
2022-03
期刊：
影响因子：
--
通讯作者：
Zhang L
中科院分区：
文献类型：
--
作者：
Xin Y;Shen C;Tang M;Guo Z;Shi Y;Gu Z;Shao J;Zhang L
N-脱甲基酶可以去除伯胺或仲胺上的甲基，进而影响生物大分子或化合物的性质和功能，但其底物范围和稳定性尚未得到系统研究。在这里，我们报告了在玫瑰色嗜热微菌肌氨酸氧化酶（TrSOX）的关键微域中的自然进化的重建，TrSOX是一种具有显著稳定性（熔化温度超过100 °C）和对映选择性的N-脱甲基酶，用于增强底物范围和对-C-N-键的催化效率。我们通过晶化和X射线衍射（XRD）对初始骨架进行了表征，得到了TrSOX的结构。关键微结构域的非保守残基的自然进化-包括催化环，辅酶口袋，底物口袋和入口位点-然后使用祖先序列重建（ASR）进行鉴定，并通过定点诱变重新创建在自然进化过程中产生的取代。单取代和双取代变体催化N-甲基-L-氨基酸的N-去甲基化分别比野生型快1800倍和6000倍。此外，这些单取代变体催化非氨基酸化合物的末端N-脱甲基化和含氮杂环中主链-C-N-键氧化为-C=N-键。值得注意的是，这些变体保留了初始骨架的对映选择性和稳定性。我们的结论是，变体的TrSOX是巨大的潜在用途在N-甲基对映体拆分，主链席夫碱的合成，生物碱的修饰或降解。
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.
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