Site-directed mutagenesis and computational study of the Y366 active site in <Emphasis Type="Italic">Bacillus subtilis</Emphasis> protoporphyrinogen oxidase |
| |
Authors: | Lu Sun Xin Wen Ying Tan Heyang Li Xing Yang Yuefang Zhao Baifan Wang Qiongyao Cao Congwei Niu Zhen Xi |
| |
Institution: | (1) State Key Laboratory of Elemento-Organic Chemistry, Department of Chemical Biology, Nankai University, 300071 Tianjin, China |
| |
Abstract: | Protoporphyrinogen IX oxidase (PPO), the last common enzyme of heme and chlorophyll biosynthesis, catalyses the oxidation
of protoporphyrinogen IX to protoporphyrin IX, with FAD as cofactor. Among PPO, Bacillus subtilis PPO (bsPPO) is unique because of its broad substrate specificity and resistance to inhibition by diphenylethers. Identification of
the activity of bsPPO would help us to understand the catalysis and resistance mechanisms. Based on the modeling and docking studies, we found
that Y366 site in bsPPO was adjacent to substrate and FAD. In order to evaluate the functional role of this site, three mutants Y366A Y366E and
Y366H were cloned and kinetically characterized. The efficiency of catalysis for Y366A and Y366H reduced to 10% of the wild-type
enzyme’s activity, while Y366E just retained 1%. Y366E shows large resistance (K
i = 153.94 μM) to acifluorfen. Molecular docking was carried out to understand the structure and functional relationship of
PPO. The experimental results from the site-directed mutagenesis are consistent with the computational studies. The residue
at position 366 is seemed to be responsible for substrate binding and catalysis and involved in herbicide resistance of bsPPO. |
| |
Keywords: | |
本文献已被 SpringerLink 等数据库收录! |
|