Yu-Cong Zhou, Sheng-Ling Fu, Hao Liu
College of Biological and Environmental Science, Zhejiang Wanli University, Ningbo, 315000, P. R. China.
Department of Pharmacy, South China Hospital, Medical School, Shenzhen University, Shenzhen, 518116, P. R. China.
Protein J. 2025 Feb;44(1):102-112. doi: 10.1007/s10930-024-10235-8. Epub 2024 Nov 2.
Previous studies reported that -amino acid oxidase (DAAO) activity was closely associated with neuropathic pain, cognitive characteristics of schizophrenia and so on. To determine DAAO mutant sites in different strains of mice and their effects on enzyme activity, we successfully constructed a prokaryotic expression system for heterologous expression of DAAO in vitro. There were total five nucleotide mutations distributed in exons 2, 8, 9, 10 of C57 mice. Three mutations located on exons 8 and 9 were synonymous mutations and had no variation on the encoded amino acid. The remaining two mutations in exons 2 (V64A) and 10 (R295H) were non-synonymous mutations, which might affect enzymatic activity and protein structure of mDAAO. Based on the determination of the kinetic constants and IC of mDAAO mutants in vitro, the differences in amino acid levels at these two sites (V64A, R295H) increased the affinity of C57 DAAO with substrate and enhanced its catalytic efficiency. Besides, the IC value of C57 DAAO was less than that of Balb/c and other DAAO mutants (SUN: reducted by about 11.9%; CBIO: reducted by about 26.5%), which meant that the affinity of C57 DAAO with CBIO was higher.
先前的研究报道，D -氨基酸氧化酶（DAAO）活性与神经性疼痛、精神分裂症的认知特征等密切相关。为了确定不同品系小鼠中DAAO的突变位点及其对酶活性的影响，我们成功构建了一个用于在体外异源表达DAAO的原核表达系统。在C57小鼠的外显子2、8、9、10中总共分布有五个核苷酸突变。位于外显子8和9上的三个突变是同义突变，编码的氨基酸没有变化。外显子2（V64A）和10（R295H）中的其余两个突变是非同义突变，这可能会影响mDAAO的酶活性和蛋白质结构。基于对mDAAO突变体体外动力学常数和IC的测定，这两个位点（V64A、R295H）氨基酸水平的差异增加了C57 DAAO与底物的亲和力并提高了其催化效率。此外，C57 DAAO的IC值小于Balb/c和其他DAAO突变体（SUN：降低约11.9%；CBIO：降低约26.5%），这意味着C57 DAAO与CBIO的亲和力更高。
