Computational Insight Into Protoporphyrinogen Ⅸ Oxidase

Protoporphyrinogen IX oxidase (PPO; EC1.3.3.4) catalyzes protoporphyrinogen IX to protoporphyrin IX. PPO is the key enzyme for the biosynthesis of protoporphyrin IX, which is the last check point of heme and chlorophyll common biosynthesis.Hence, PPO has been identified as one of the most importance action targets for the treatment of some important diseases including cancer and variegated porphyria (VP). In the agriculture, PPO inhibitors have been used as herbicides for many years. Inhibition of PPO in plants leads the accumulation of protogen, which exports to the cytoplasm and forms porphyrm through nonenzymatic oxidation. The photosensitizing porphyrin can produce singlet oxygen which can cause peroxidation of membrane lipids and cell death. PPO inhibitors herbicides are highly effective, low toxic to human and friendly to environment. The development of the new PPO inhibitors herbicides is the hot topic of the field. In humans, defects in the PPO gene, resulting in approximately50%decreased activity of PPO, is responsible for the dominantly inherited disorder VP. VP is a type of acute hepatic porphyria, which is characterized by an abnormal pattern of porphyrin excretion. VP has been found worldwide, and is particularly prevalent in the white population of South Africa.Since PPO is essential for the biological process, and has widely application in the agricultural field and medicine, we chose PPO as research object. We hope to gain understanding about the molecular mechanism of the PPO. In this dissertation we focus primary on the following aspects:1. Applying bioinformatics studies on PPO. We had identified the conserved and noconserved residues of the active site of PPO from different species by sequence alignments. We studied the contributions of these to the properties of PPO from different species, which provided information for the site directed mutagenesis study on PPO. We compared the structural domains of PPO to reveal the sequence and structural difference contribution to the location and assembly of PPO from different species. These studies provided important information for the further studies on the difference on the property of PPO and the relationship between structure and function of PPO.2. Applying studies on the interaction between substrate and PPO. We docked substrate into PPO from tobacco mitochondria, human and Bacillus subtilis. Molecular dynamics simulation were then performed on docked substrate-PPO complex. We access the the correct substrate-PPO binding models based on the molecular dynamics simulation results, with the site directed mutagenesis studies and quantum mechanics calculation. We also compared the binding mode of substrate in PPO from different species, in which we identified the origin of the difference catalytic activity on PPOs.3. Applying studies on the function of PPO based on structure. In this part of work, we applied site directed mutagenesis and computational studies on PPO, which including:(1) Study on the active site residues of PPO. We combined the site directed mutagenesis and molecular dynamics simulation to identified the function of the residues of human PPO.(2) Study on the residues cluster which regulate the activity of PPO. We applied structural biology, biochemical and simulation studies to identify a polar interaction network surrounding Arg59and Asp65in human and Bacillus subtilis PPO which can regulate the activity of PPO. This interaction network help to stabilize the environment of the isoalloxazine ring of FAD, in which stabilize the interaction between substrate and FAD to maintain the activity of PPO. By compared the sequence and structure of PPO, we discover this interaction network in conserved in PPO from different species.4. Applying studies on the molecular mechanism of the variegated porphyria. We applied structural study on the variegated porphyria, which indicated that the44VP-causing mutants distributed on all of the three structure domains of human PPO. We categorized these mutants into5potential categories, i.e., affecting the substrate binding, FAD binding, hydrophobic core, secondary structure and surface of human PPO. We then combined molecular dynamics simulation and statistical study to quantitative insight into the molecular mechanism of VP. In light of the collision theory, we develop a descriptor which can quantitative describe the catalytic activity of PPO, which called probability of privileged conformations. We successfully predicted the catalytic activity of44VP-causing mutants by calculating their probability of privileged conformations, and we elucidated the detailed molecular mechanism of these mutants. Based on these results we develop a protocol which can predict the catalytic activity of PPO. This protocol can be used to predict the activity of human PPO mutants and other species of PPO.5. Applying studies on the interaction between PPO and inhibitors. We applied structural biology study and computational study on the interaction between PPO and inhibitors, which mainly on:(1) The crystal structures of the complex of PPO and inhibitor. We obtained the crystal structures of human PPO and the inhibitors, oxyfluorfen and fomesafen, in which the resolution was2.3A and2.0A, respectively. We compared the binding mode of the acifluorofen, oxyfluorfen and fomesafen to human PPO and their activity, in which we found that the Arg97in human PPO is a important binding site for inhibitors.(2) The selectivity to inhibitor in PPO from different species. We performed molecular dynamics simulation and MM-GBSA energy calculation on the complex of acifluorofen and PPO from tobacco mitochondria, human and Bacillus subtilis. We found that, the residues Phe392, Leu372, Leu356, Phe353and Arg98is the "hot spots" for the selectivity to inhibitor of PPO. PPO regulated the binding of inhibitors by altering the size and electro-property of the side chain of these residues, which was validated by the mutagenesis study.In total, we combined the biochemical and computational methods to study the molecular mechanism of PPO. We identified the differences in the sequence and structure of PPO, which provides guidance for the study of the property of PPO and mutagenesis experiments. We obtained the binding mode of substrate in PPO from different species, which help to elucidate the catalytic mechanism of PPO and provide information for the design of new PPO inhibitor. We applied study on the mutant of PPO, which help to understand the relationship of the structure and function of PPO. We developed a protocol to quantitative predict the catalytic of VP-causing mutants, and make detailed description on the molecular mechanism of VP. And we also elucidated the selectivity of PPO, which provides the structural basis for the design of PPO inhibitor.