Correlation Analysis of Key Residue Sites between Computational-Aided Design Thermostability d-Amino Acid Oxidase and Ancestral Enzymes

热稳定性 蛋白质设计 蛋白质工程 氨基酸 D 生物催化 生物化学 热稳定性 化学 同系序列 酶动力学 氧化酶试验 蛋白质超家族 立体化学 蛋白质结构 活动站点 催化作用 有机化学 基因 反应机理
作者
Liu‐Yu Wang,Heng Tang,Jin‐Qiao Zhao,Meng‐Nan Wang,Ya‐Ping Xue,Yu‐Guo Zheng
出处
期刊:Journal of Agricultural and Food Chemistry [American Chemical Society]
卷期号:71 (50): 20177-20186 被引量:5
标识
DOI:10.1021/acs.jafc.3c06865
摘要

The d-amino acid oxidase (DAAO) from Rhodotorula taiwanensis has proven to have great potential for applications due to its excellent catalytic kinetic parameters. However, its poor thermal stability has limited its performance in biocatalysis. Herein, starting from the variant SHVG of RtwDAAO, this study employed a comprehensive computational design approach for protein stability engineering, resulting in positive substitutions at specific sites (A43S, T45M, C234L, E195Y). The generated variant combination, SHVG/SMLY, exhibited a significant synergistic effect, leading to an extension of the half-life and Tmapp. The ancestral sequence reconstruction revealed the conservation of the variant sites. The association of the variant sites with the highly stable ancestral enzyme was further explored. After determining the contribution of the variant sites to thermal stability, it was applied to other homologous sequences and validated. Molecular dynamics simulations indicated that the increased hydrophobicity of the variant SHVG/SMLY was a key factor for the increased stability, with strengthened intersubunit interactions playing an important role. In addition, the physical properties of the amino acids themselves were identified as crucial factors for thermal stability generality in homologous enzymes, which is important for the rapid acquisition of a series of stable enzymes.
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