Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Animals
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Catalysis
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Cloning, Molecular
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Dimethylglycine Dehydrogenase / chemistry
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Dimethylglycine Dehydrogenase / genetics
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Dimethylglycine Dehydrogenase / isolation & purification*
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Dimethylglycine Dehydrogenase / metabolism*
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Flavin-Adenine Dinucleotide / metabolism*
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Gene Expression
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Mass Spectrometry
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Mitochondria / enzymology*
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Mitochondrial Proteins / chemistry
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Mitochondrial Proteins / genetics
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Mitochondrial Proteins / isolation & purification*
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Mitochondrial Proteins / metabolism*
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Molecular Sequence Data
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Rats
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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Recombinant Proteins / isolation & purification
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Recombinant Proteins / metabolism
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Spectrophotometry
Substances
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Mitochondrial Proteins
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Recombinant Proteins
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Flavin-Adenine Dinucleotide
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Dimethylglycine Dehydrogenase
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Dmgdh protein, rat