Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: KEGG:D02011 (FAD)
 5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Protoporphyrinogen oxidase (EC 1-3-3-4), the 60-kDa membrane-bound flavoenzyme that catalyzes the final reaction of the common branch of the heme and chlorophyll biosynthesis pathways in plants, is the molecular target of diphenyl ether-type herbicides. It is highly resistant to proteases (trypsin, endoproteinase Glu-C, or carboxypeptidases A, B, and Y), because the protein is folded into an extremely compact form. Trypsin maps of the native purified and membrane-bound yeast protoporphyrinogen oxidase show that this basic enzyme (pI > 8.5) was cleaved at a single site under nondenaturing conditions, generating two peptides with relative molecular masses of 30,000 and 35,000. The endoproteinase Glu-C also cleaved the protein into two peptides with similar masses, and there was no additional cleavage site under mild denaturing conditions. N-terminal peptide sequence analysis of the proteolytic (trypsin and endoproteinase Glu-C) peptides showed that both cleavage sites were located in putative connecting loop between the N-terminal domain (25 kDa) with the betaalphabeta ADP-binding fold and the C-terminal domain (35 kDa), which possibly is involved in the binding of the isoalloxazine moiety of the FAD cofactor. The peptides remained strongly associated and fully active with the Km for protoporphyrinogen and the Ki for various inhibitors, diphenyl-ethers, or diphenyleneiodonium derivatives, identical to those measured for the native enzyme. However, the enzyme activity of the peptides was much more susceptible to thermal denaturation than that of the native protein. Only the C-terminal domain of protoporphyrinogen oxidase was labeled specifically in active site-directed photoaffinity-labeling experiments. Trypsin may have caused intramolecular transfer of the labeled group to reactive components of the N-terminal domain, resulting in nonspecific labeling. We suggest that the active site of protoporphyrinogen oxidase is in the C-terminal domain of the protein, at the interface between the C- and N-terminal domains.
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PMID:The domain structure of protoporphyrinogen oxidase, the molecular target of diphenyl ether-type herbicides. 972 41

Propionibacterium acnes strain ATCC 6919 catalyzes the isomerization of the double bond at the C9 position in linoleic acid (c9,c12, 18:2) to form t10,c12 conjugated linoleic acid (CLA, 18:2). CLA has significant health benefits in animal and human. The linoleic acid C9 isomerase was purified to an apparent homogeneity by successive chromatography on diethylaminoethyl (DEAE) anion exchange, hydrophobic interaction, and chromatofocusing columns. Two degenerated oligonucleotide primers were synthesized according to the N-terminal peptide sequence to clone, by polymerase chain reaction (PCR), a short nucleotide sequence (62 bp) of the isomerase gene. The linoleic acid isomerase gene (lai) was subsequently cloned by inverse PCR. The amino acid sequence deduced from the lai coding sequence predicts a protein of 424 amino acid residues (48 kDa), excluding the N-terminal methionine, which was absent in the polypeptide purified from the native host. The isomerase shares no significant sequence homology to other enzymes except a flavin-binding domain in the N-terminal region. The recombinant isomerase purified from Escherichia coli showed a typical ultraviolet spectrum for FAD-bound proteins. The recombinant enzyme produced a single isomer of t10,c12-CLA from linoleic acid, as demonstrated by gas chromatography and gas chromatography-mass spectrum analysis. The recombinant isomerase protein was expressed at high levels in E. coli, but it was almost totally sequestered in inclusion bodies. The level of active isomerase was increased 376-fold by medium and process optimization in bench-scale fermentors.
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PMID:Linoleic acid isomerase from Propionibacterium acnes: purification, characterization, molecular cloning, and heterologous expression. 1805 48

Renalase is a recently discovered secretory protein involved in the regulation of blood pressure. Cells synthesize all known isoforms of human renalase (1 and 2) as flavoproteins. Accommodation of FAD in the renalase protein requires the presence of its N-terminal peptide. However, in secretory proteins, such peptides are usually cleaved during their export from the cell. In the present study, we have isolated human renalase from urinary samples of healthy volunteers and human recombinant renalases 1 and 2 expressed in Escherichia coli cells. In these proteins, we investigated the presence of the renalase N-terminal peptide and the FAD cofactor and performed computer-aided molecular analysis of the renalase crystal structure to evaluate possible consequences of removal of the N-terminal peptide. In contrast to human recombinant renalase isoforms 1 and 2 containing non-covalently bound FAD and clearly detectable N-terminal peptide, renalase purified from human urine lacks both the N-terminal signal peptide and FAD. The computer-aided analysis indicates that the removal of this peptide results in inability of the truncated renalase to bind the FAD cofactor. Thus, our results indicate that human renalase secreted in urine lacks its N-terminal peptide, and therefore catalytic activities of urinary renalase reported in the literature cannot be attributed to FAD-dependent mechanisms. We suggest that FAD-dependent catalytic functions are intrinsic properties of intracellular renalases, whereas extracellular renalases act in FAD- and possibly catalytic-independent manner.
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PMID:Human urinary renalase lacks the N-terminal signal peptide crucial for accommodation of its FAD cofactor. 2591 Jun 47