The purified recombinant precursor of rat mitochondrial dimethylglycine dehydrogenase binds FAD via an autocatalytic reaction.
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.
Brizio
Carmen
C
Dipartimento di Biochimica e Biologia Molecolare E. Quagliariello, Università degli Studi di Bari, Via Orabona 4, 70126 Bari, Italy.
Brandsch
Roderich
R
Douka
Maria
M
Wait
Robin
R
Barile
Maria
M
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
D024101:Mitochondrial Proteins; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D050893:Dimethylglycine Dehydrogenase; C494400:Dmgdh protein, rat