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Query: KEGG:D02011 (FAD)
 5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Protoporphyrinogen oxidase (E.C.1.3.3.4) (PPO) catalyzes the penultimate step in the heme biosynthetic pathway. Deficiency in activity of this enzyme results in the human genetic disease variegate porphyria. Herein we detail the cloning, expression, purification and characterization of the normal and variegate porphyria forms of human PPO. The cDNA sequence for human ppo is approximately 1.8 kb in length and codes for a protein of 477 amino acids. This protein, which does not contain a typical cleavable mitochondrial targeting sequence, is approximately 51 kDa and contains a putative dinucleotide binding motif near the amino terminus. The active enzyme is a homodimer and contains an FAD. Attachment of a six his amino terminal tag allows for the rapid and efficient purification of approximately 10 mg of enzyme from one liter of E. coli culture. Three variegate porphyria mutant PPO enzymes were expressed and characterized. These mutations, R59W, R168C and A433P, result in decreased enzyme activity by causing a decrease in kcat without a significant change in Km for the substrate protoporphyrinogen IX. Purified R59W lacks the FAD cofactor which may be explained by the fact that this mutation resides within the dinucleotide binding motif of PPO.
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PMID:Characteristics of human protoporphyrinogen oxidase in controls and variegate porphyrias. 907 90

Protoporphyrinogen IX oxidase (protox) catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX in the penultimate step of heme and chlorophyll biosynthesis in animals and plants. Protox is the target of light-dependent peroxidizing herbicides and is inhibited at nanomolar levels by several chemical classes including tetrahydrophthalimides (discussed below) and diphenyl ethers (e.g., acifluorfen) usually with little selectivity between the mammalian and plant enzymes. The herbicide binding site is examined here with a photoaffinity radioligand optimized on the basis of structure-activity relationships. A radiosynthetic procedure is described for this new herbicidal probe, N-(5-azido-4-chloro-2-fluorophenyl)-3,4,5, 6-[3H]tetrahydrophthalimide ([3H]AzTHP), resulting in high specific activity (2.6 TBq/mmol). Human protox expressed in Escherichia coli and purified by affinity chromatography is used with [3H]AzTHP to characterize the herbicide/substrate binding site. Specific binding of [3H]AzTHP to human protox is rapid, completely reversible in the absence of light with a Kd of 93 nM, and competitively inhibited by the 5-propargyloxy analogue and by acifluorfen, which are known to bind at the substrate (protoporphyrinogen) site. The Bmax establishes one [3H]AzTHP binding site per FAD. Diphenyleneiodonium, proposed to inhibit protox by interaction with the FAD cofactor, inhibits enzyme activity by 48% at 100 micro M without affecting [3H]AzTHP binding in the presence or absence of substrate, suggesting that the herbicide binding site may not be proximal to FAD. The first step has been taken in photoaffinity labeling the herbicide/substrate site with [3H]AzTHP resulting in apparent covalent derivatization of 13% of the herbicide binding site.
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PMID:Human protoporphyrinogen oxidase: relation between the herbicide binding site and the flavin cofactor. 957 77

Protoporphyrinogen IX oxidase (PPO), the last common enzyme of haem and chlorophyll biosynthesis, catalyses the oxidation of protoporphyrinogen IX to protoporphyrin IX. The membrane-embedded flavoprotein is the target of a large class of herbicides. In humans, a defect in PPO is responsible for the dominantly inherited disease variegate porphyria. Here we present the crystal structure of mitochondrial PPO from tobacco complexed with a phenyl-pyrazol inhibitor. PPO forms a loosely associated dimer and folds into an FAD-binding domain of the p-hydroxybenzoate-hydrolase fold and a substrate-binding domain that enclose a narrow active site cavity beneath the FAD and an alpha-helical membrane-binding domain. The active site architecture suggests a specific substrate-binding mode compatible with the unusual six-electron oxidation. The membrane-binding domains can be docked onto the dimeric structure of human ferrochelatase, the next enzyme in haem biosynthesis, embedded in the opposite side of the membrane. This modelled transmembrane complex provides a structural explanation for the uncoupling of haem biosynthesis observed in variegate porphyria patients and in plants after inhibiting PPO.
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PMID:Crystal structure of protoporphyrinogen IX oxidase: a key enzyme in haem and chlorophyll biosynthesis. 1505 73

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.
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PMID:Site-directed mutagenesis and computational study of the Y366 active site in Bacillus subtilis protoporphyrinogen oxidase. 1926 55

Protoporphyrinogen IX oxidase (PPO) converts protoporphyrinogen IX to protoporphyrin IX, playing an important part in the heme/chlorophyll biosynthetic pathway. Bacillus subtilis PPO (bsPPO) is unique among PPO family members in that it is a soluble monomer, is inefficiently inhibited by the herbicide acifluorfen (AF) and has broader substrate specificity than other PPO enzymes. Here, we present the crystal structure of bsPPO bound to AF. Our structure shows that the AF molecule binds to a new site outside the previously identified inhibitor binding pocket. Most importantly, the benzene ring of the 2-nitrobenzoic acid moiety of AF lies parallel to the isoalloxazine ring of FAD at a distance of less than 3.5A, providing a framework for the interaction of FAD with the substrate protoporphyrinogen IX. Furthermore, our structure reveals that the larger substrate binding chamber and predominantly positively charged chamber surface of bsPPO are more favorable for the binding of coproporphyrinogen-III. These crystallographic findings uncover biochemically unique properties of bsPPO, providing important information for further understanding the enzymatic mechanism.
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PMID:Structural insight into unique properties of protoporphyrinogen oxidase from Bacillus subtilis. 1994 66