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低温EM结构和超高效的 ochratoxin A-detoxifying amidohydrolase 的合理工程

Longhai Dai¹, Du Niu¹, Jian-Wen Huang¹

¹State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Hongshan Laboratory, Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan 430062, PR China.

Journal of hazardous materials

|June 18, 2023

概括

这项研究揭示了Stenotrophomonas acidaminiphila amidohydrolase 3 (ADH3) 对于Ochratoxin A (OTA) 排毒的催化机制. 工程ADH3增强了它的活性,为改进的真菌毒素降解策略铺平了道路.

科学领域:

生物化学生物化学
结构生物学结构生物学
酶学是一种酶学.

背景情况:

毒素A (OTA) 是农产品中普遍存在的真菌毒素,对人类和动物健康构成风险.
酶解毒为缓解OTA污染提供了一个有希望的策略.
来自Stenotrophomonas acidaminiphila的阿米多酶3 (ADH3) 是一种高效的酶,用于将OTA化成无毒化合物.

研究的目的:

通过结构和生化分析阐明ADH3的催化机制.
研究关键残留物在OTA结合和水解中的作用.
为增强催化活性设计ADH3并探索其生产可行性.

主要方法:

单粒子冷电子显微镜 (cryo-EM) 用于确定ADH3在不同状态中的结构.
局部定向突变发生和生物化学试验,以分析酶功能.
在微生物宿主 (大肠杆菌和Pichia pastoris) 中,ADH3及其变体的异质表达.

主要成果:

在2.5-2.7 Å分辨率下解析了阿波,Phe和OTA结合的ADH3的冷EM结构.
突变和生物化学分析确定了涉及OTA结合的关键残留物.
工程变体S88E的催化活性增加了3.7倍,具有对改善相互作用的结构见解.
在Pichia pastoris表达时,ADH3变异S88E表现出类似的活性,表明工业生产潜力.

结论:

这项研究为ADH3介导的Ochratoxin A降解机制提供了详细的结构洞察力.
合理设计ADH3可以显著提高其真菌毒素排毒效率.
使用Pichia pastoris用于ADH3生产的可行性为毒素控制中的实际应用开辟了道路.

关键词:

氨基基基酶氨基基酶.催化效率催化效率催化效率催化机制是一种催化机制.化-EM结构的结构.奥克拉托克辛A是一种蛋白质工程是一种蛋白质工程.

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