Site-directed mutagenesis and computational study of the Y366 active site in Bacillus subtilis protoporphyrinogen oxidase.
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 microM) 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.
Sun
Lu
L
Department of Chemical Biology, Nankai University, Tianjin, China.
Wen
Xin
X
Tan
Ying
Y
Li
Heyang
H
Yang
Xing
X
Zhao
Yuefang
Y
Wang
Baifan
B
Cao
Qiongyao
Q
Niu
Congwei
C
Xi
Zhen
Z
D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D055162:Biocatalysis; D020134:Catalytic Domain / Q000235:genetics*; D002942:Circular Dichroism; D019295:Computational Biology / Q000379:methods*; D003198:Computer Simulation; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000096:biosynthesis / Q000378:metabolism; D009579:Nitrobenzoates / Q000378:metabolism / Q000494:pharmacology; D010084:Oxidation-Reduction; D011485:Protein Binding; D050768:Protoporphyrinogen Oxidase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011524:Protoporphyrins / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism
D004791:Enzyme Inhibitors; D050505:Mutant Proteins; D009579:Nitrobenzoates; D011524:Protoporphyrins; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; C028025:protoporphyrin IX; D050768:Protoporphyrinogen Oxidase; C018425:acifluorfen