title: The purified recombinant precursor of rat mitochondrial dimethylglycine dehydrogenase binds FAD via an autocatalytic reaction.
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.
authors.affiliation: Dipartimento di Biochimica e Biologia Molecolare E. Quagliariello, Università degli Studi di Bari, Via Orabona 4, 70126 Bari, Italy.
authors.forename: Carmen
authors.initials: C
authors.lastname: Brizio
authors.forename: Roderich
authors.initials: R
authors.lastname: Brandsch
authors.forename: Maria
authors.initials: M
authors.lastname: Douka
authors.forename: Robin
authors.initials: R
authors.lastname: Wait
authors.forename: Maria
authors.initials: M
authors.lastname: Barile
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D003001:Cloning, Molecular; D050893:Dimethylglycine Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D015870:Gene Expression; D013058:Mass Spectrometry; D008928:Mitochondria / Q000201:enzymology*; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D008969:Molecular Sequence Data; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013053:Spectrophotometry
chemical_list: D024101:Mitochondrial Proteins; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D050893:Dimethylglycine Dehydrogenase; C494400:Dmgdh protein, rat