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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志  2015, Vol. 35 Issue (8): 68-75    DOI: 10.13523/j.cb.20150810
研究报告     
6-羟基-3-琥珀酰吡啶单加氧酶HspB的结构研究
李鹏鹏1, 于浩1,2, 许平1, 唐鸿志1
1. 上海交通大学生命科学技术学院 微生物代谢国家重点实验室 上海 200240;
2. 青岛农业大学生命科学学院 青岛 266109
The Structural Studies of 6-Hydroxy-3-succinoyl-pyridine Monooxygenase
LI Peng-peng1, YU Hao1,2, XU Ping1, TANG Hong-zhi1
1. State Key Laboratory of Microbial Metabolism, School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China;
2. Collage of Life Science, Qingdao Agriculture University, Qingdao 266109, China
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摘要:

在蛋白晶体结构难以获得的情况下,通过设计突变体来获取6-羟基-3-琥珀酰吡啶单加氧酶HspB的结构信息。首先获取HspB蛋白的同源序列并进行比对,之后对HspB蛋白进行同源建模和从头建模,并与底物2,5-二羟基吡啶(HSP)进行对接模拟;通过分子模拟、序列比对和参考同源蛋白晶体三种方式,设计并构建HspB酶的25个突变体;通过突变体的表达纯化和酶动力学常数测定来研究HspB的结构性质。根据实验结果,推测FAD的正确结合在稳定HspB蛋白结构中具有重要的作用,同时推测底物HSP和辅酶NADH处于同一活性中心并与不同位点相互作用。吡啶衍生物是极具工业价值的化合物,生物催化法是合成吡啶衍生物的有效途径,而吡啶衍生物的生物催化研究较少,通过考察突变体的性质,推测了HspB的部分结构信息,为此类吡啶单加氧酶的工业改造和应用奠定了基础。

关键词: HspB突变体结构信息酶动力学常数    
Abstract:

Pyridine derivatives are the important value-added chemicals, and biocatalysis is a potential technology for the industrial synthesis of pyridine derivatives. The structure information of 6-hydroxy-3-succinoyl-pyridine monooxygenase (HspB) by mutant construction was investigated. The space structure of HspB has been built through computer modeling and docked with its substrate HSP(2,5-dihydroxy-pyridine). Then 25 mutations of HspB were constructed and studied, according to molecular simulation, sequence alignment, and homologous crystal references. All the mutants have been expressed and purified, and the kinetic parameters of soluble mutants have been measured. According to the properties of mutants, it can infer that the correct binding of FAD possesses important roles in protein stability, and moreover, substrate HSP, and co-enzyme NADH live in the same activity center in HSP but interact with different amino acids. The structure information will help us for the industrial application of pyridine monooxygenase.

Key words: HspB    Mutation    Structure information    Kinetic parameter
收稿日期: 2015-03-13 出版日期: 2015-08-25
ZTFLH:  Q816  
基金资助:

国家自然科学基金(31422004,31270154,31230002)、上海市青年科技启明星项目(13QA1401700)资助项目

通讯作者: 唐鸿志     E-mail: tanghongzhi@sjtu.edu.cn
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引用本文:

李鹏鹏, 于浩, 许平, 唐鸿志. 6-羟基-3-琥珀酰吡啶单加氧酶HspB的结构研究[J]. 中国生物工程杂志, 2015, 35(8): 68-75.

LI Peng-peng, YU Hao, XU Ping, TANG Hong-zhi . The Structural Studies of 6-Hydroxy-3-succinoyl-pyridine Monooxygenase. China Biotechnology, 2015, 35(8): 68-75.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20150810        https://manu60.magtech.com.cn/biotech/CN/Y2015/V35/I8/68


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