The purified recombinant precursor of rat mitochondrial dimethylglycine dehydrogenase binds FAD via an autocatalytic reaction

Int J Biol Macromol. 2008 Jun 1;42(5):455-62. doi: 10.1016/j.ijbiomac.2008.03.001. Epub 2008 Mar 13.

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

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Catalysis
  • Cloning, Molecular
  • Dimethylglycine Dehydrogenase / chemistry
  • Dimethylglycine Dehydrogenase / genetics
  • Dimethylglycine Dehydrogenase / isolation & purification*
  • Dimethylglycine Dehydrogenase / metabolism*
  • Flavin-Adenine Dinucleotide / metabolism*
  • Gene Expression
  • Mass Spectrometry
  • Mitochondria / enzymology*
  • Mitochondrial Proteins / chemistry
  • Mitochondrial Proteins / genetics
  • Mitochondrial Proteins / isolation & purification*
  • Mitochondrial Proteins / metabolism*
  • Molecular Sequence Data
  • Rats
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Spectrophotometry

Substances

  • Mitochondrial Proteins
  • Recombinant Proteins
  • Flavin-Adenine Dinucleotide
  • Dimethylglycine Dehydrogenase
  • Dmgdh protein, rat