Studies On Mechanism And Inhibition Kinetics Of Protoporphyrinogen Oxidase

Protoporphyrinogen oxidase (PPO; EC 1.3.3.4) catalyses the oxygen-dependent aromatization of protoporphyrinogen IX to the fully conjugated macrocycle of protoporphyrin IX, which widely exists in animals, plants, fungi and bacteria. It is the last common step of tetrapyrrole biosynthesis for the formation of haems and chlorophylls.PPO locates in the chloroplasts and mitochondria, for the synthesis of chlorophyll and heme, plays an important role, which determines the great significance in the life system. In humans, the PPO defects can cause autosomal dominant disease Variegated Porphyria (VP). In the agricultural field, PPO inhibitors have been used as herbicides for many years. They appear to be ideal herbicides, because they act rapidly with low use-rate and do not harm mammals under normal conditions, as well as to be environmental friendly and low level of resistance as competitive inhibitors. But because of long for forty years in use, natural occurrence of weed resistance to PPO inhibitors has been reported since 2004 and it has apparently begun to expend. So it is necessary to find alternative inhibitors with more potency, especially that of novel scaffolds to solve the antiresistance problem and limitations of commercial inhibitors.In this paper, we hope to obtain a full range of information about protoporphyrinogen oxidase, and we mainly carried out the work in the following four areas:Firstly, we crystallized human protoporphyrinogen oxidase (hPPO) with the inhibitor acifluorfen (AF) through a variety of protein crystals to explore the optimizational culture conditions for hPPO, we got the crystal structure of hPPO in complex with the cofactor flavin adenine dinucleotide (FAD) and the inhibitor AF at a resolution of 1.9 A. The structural and biochemical analyses revealed the molecular details of FAD and AF binding to hPPO. Structural analysis and gel chromatography indicated that hPPO is a monomer rather than a homodimer in vitro.Secondly, because efforts to crystallize the substrate or substrate analog with hPPO failed, we performed in silico docking of the substrateprotoporphyrinogen IX into the crystal structure of hPPO to find the significant residues contributing to PPO activity, and verified the importance of these residues.Thirdly, We studied the role of electron acceptor O2 in the catalytical reaction, and used stopped-flow method to measure the rate constant of the enzyme reaction. we also proposed new insights for the mechanism.Fourthly, we studied the inhibitor kinetics by high-throughput screening to design the new and efficient inhibitor molecules rationally.In summary, this paper presented the perspective of structural biology of hPPO. The interactions between the enzyme and substrate/inhibitor/cofactor in protoporphyrinogen oxidase were studied by mutagenesis, kinetics and computational methods. The inhibitor resistance was also be studied by high-throughput screening. All of these results would shed light on the research of inhibitor or regulator molecular exploitation and design, as well as the variegated porphyria research..