TY - JOUR
T1 - Site-directed mutagenesis and computational study of the Y366 active site in Bacillus subtilis protoporphyrinogen oxidase
AU - Sun, Lu
AU - Wen, Xin
AU - Tan, Ying
AU - Li, Heyang
AU - Yang, Xing
AU - Zhao, Yuefang
AU - Wang, Baifan
AU - Cao, Qiongyao
AU - Niu, Congwei
AU - Xi, Zhen
N1 - Funding Information:
This work was supported by the National Key Project for Basic Research of China (2003CB114403), National Natural Science Foundation of China (20432010, 20572053, 20421202, 90713011), Natural Science Foundation of Tianjin (06TXTJJC14100) and Nankai University ISC. We also thank referees of this paper for many critical discussions.
PY - 2009/9
Y1 - 2009/9
N2 - 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.
AB - 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.
KW - Kinetics
KW - Molecular docking
KW - Protoporphyrinogen oxidase
KW - Site-directed mutagenesis
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U2 - 10.1007/s00726-009-0256-5
DO - 10.1007/s00726-009-0256-5
M3 - Article
C2 - 19266155
AN - SCOPUS:69249233521
SN - 0939-4451
VL - 37
SP - 523
EP - 530
JO - Amino Acids
JF - Amino Acids
IS - 3
ER -