Flavinylation of the precursor of mitochondrial dimethylglycine dehydrogenase by intact and solubilised mitochondria

Abstract

The £avinylation 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 £avinylation of pMe 2 -GlyDH was strictly dependent on the addition of mitochondrial protein(s), among which the mitochondrial £avinylation 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 £avinylation to proceed. The comparison of the time course of the £avinylation and the processing of pMe 2 GlyDH demonstrated that the covalent attachment of the £avin moiety preceded the presequence processing by mitochondrial processing peptidase. ß

Riboflavin Uptake and FAD Synthesis in Saccharomyces cerevisiae Mitochondria

“…Indeed, a number of mitochondrial apo-flavoenzymes have been reported to be charged with their cofactors only inside the organelle (5,6,34,35). The experiments described here demonstrate that Flx1p plays a crucial role in maintaining a normal level of FAD-binding enzyme activity.…”

“…FAD synthesis occurs inside the organelle from imported Rf and mitochondrial ATP, consistent with the presence of a mitochondrial riboflavin kinase (EC 2.7.1.26) and an FAD synthetase (EC 2.7.7.2) (3,4). Newly synthesized FAD can be either efficiently incorporated into newly imported apo-flavoproteins (5,6) or can be exported into the outer mitochondrial compartments, where it is reconverted to Rf by FAD pyrophosphatase (EC 3.6.1.18) and FMN phosphohydrolase (EC 3.1.3.2) in a recycling pathway, i.e. the Rf-FAD cycle (4,7).…”

Coordinated and reversible reduction of enzymes involved in terminal oxidative metabolism in skeletal muscle mitochondria from a riboflavin‐responsive, multiple acyl‐CoA dehydrogenase deficiency patient

“…A riboflavin recycling pathway has also been demonstrated in rat liver mitochondria (the riboflavin-FAD cycle) [1,2]. Several lines of evidence suggest that FAD synthesis [2,3] and binding to apo-enzymes occur inside mitochondria [4]; covalent flavinylation (for a review, see [5,6]) appears to be accelerated by chaperons or specific intramitochondrial proteins [7][8][9].…”

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

“…The plasmid pET-21b(+)-pMe 2 GlyDH was constructed starting from pSPT19-pMe 2 GlyDH plasmid, which contains the pMe 2 GlyDH cDNA sequence in the vector pSPT19 [34,36]. PCR was used to introduce a NheI restriction site at the 5 end of the pMe 2 GlyDH coding sequence in front of the Leu-2 codon (CTC), delete the stop codon, and introduce an EagI site at the 3 end of the pMe 2 GlyDH coding sequence.…”

“…Both the mature (mMe 2 GlyDH) and the precursor form of Me 2 GlyDH (pMe 2 GlyDH) were synthesized in the RL system in their apoenzyme forms; folding of both the mature and the precursor apo-form into the trypsin-resistant holoenzyme proceeded spontaneously in the presence of FAD, in line with an autocatalytic process [34]. Mitochondrial protein factors, which are still unidentified, are able to stimulate holoenzyme formation [35,36].…”