Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.64 (MPP)
 1,876 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We analyzed the folding, covalent flavinylation, and mitochondrial import of the rabbit reticulocyte lysate-translated bacterial 6-hydroxy-D-nicotine oxidase (6-HDNO) fused to the mitochondrial targeting sequence of rat liver dimethylglycine dehydrogenase. Translation of 6-HDNO in FAD-supplemented reticulocyte lysate resulted in a protein that contained covalently incorporated FAD, exhibited enzyme activity, and was trypsin-resistant, a characteristic of the tight conformation of the holoenzyme. The attached mitochondrial presequence did not prevent folding, binding of FAD, or enzyme activity of the 6-HDNO moiety of the fusion protein (pre-6-HDNO). Pre-6-HDNO was imported into rat liver mitochondria and processed by the mitochondrial processing peptidase. Incubation of the trypsin-resistant pre-holo-6-HDNO protein with deenergized rat liver mitochondria demonstrated that upon contact with mitochondria, the protein was unfolded and became trypsin sensitive. Mitochondrial import assays showed that the unfolded pre-holo-6-HDNO with covalently attached FAD was imported into rat liver mitochondria. Inside the mitochondrion the holo-6-HDNO was refolded into the trypsin-resistant conformation. However, when pre-apo-6-HDNO was imported only part of the protein became trypsin resistant (approximately 20%). Addition of FAD and the allosteric effector glycerol 3-phosphate to apo-6-HDNO containing mitochondrial matrix was required to transform the protein into the trypsin-resistant conformation characteristic of holo-6-HDNO.
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PMID:Folding, flavinylation, and mitochondrial import of 6-hydroxy-D-nicotine oxidase fused to the presequence of rat dimethylglycine dehydrogenase. 771 2

The flavinylation and the presequence processing of the mitochondrial matrix enzyme dimethylglycine dehydrogenase (Me(2)GlyDH) were investigated with the reticulocyte lysate translated precursor (pMe(2)GlyDH) added to solubilised mitoplasts of rat liver mitochondria. The flavinylation of pMe(2)GlyDH was strictly dependent on the addition of mitochondrial protein(s), among which the mitochondrial flavinylation stimulating factor [Brizio C., et al. (2000) Eur. J. Biochem 267, 4346-4354], that actively promotes holo-Me(2)GlyDH formation. The precursor processing, that accompanies the biogenesis of the enzyme, was not required to allow the flavinylation to proceed. The comparison of the time course of the flavinylation and the processing of pMe(2)GlyDH demonstrated that the covalent attachment of the flavin moiety preceded the presequence processing by mitochondrial processing peptidase.
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PMID:Flavinylation of the precursor of mitochondrial dimethylglycine dehydrogenase by intact and solubilised mitochondria. 1209 34

The precursor of the rat mitochondrial flavoenzyme dimethylglycine dehydrogenase (Me(2)GlyDH) has been produced in Escherichia coli as a C-terminally 6-His-tagged fusion protein, purified by one-step affinity chromatography and identified by ESI-MS/MS. It was correctly processed into its mature form upon incubation with solubilized rat liver mitoplasts. The purified precursor was mainly in its apo-form as demonstrated by immunological and fluorimetric detection of covalently bound flavin adenine dinucleotide (FAD). Results described here definitively demonstrate that: (i) covalent attachment of FAD to Me(2)GlyDH apoenzyme can proceed in vitro autocatalytically, without third reactants; (ii) the removal of mitochondrial presequence by mitochondrial processing peptidase is not required for covalent autoflavinylation.
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PMID:The purified recombinant precursor of rat mitochondrial dimethylglycine dehydrogenase binds FAD via an autocatalytic reaction. 1842 46