Folding, flavinylation, and mitochondrial import of 6-hydroxy-D-nicotine oxidase fused to the presequence of rat dimethylglycine dehydrogenase.
We analyzed the folding, covalent flavinylation, and mitochondrial import of the rabbit reticulocyte lysate-translated bacterial 6-hydroxy-D-nicotine oxidase (6-HDNO) fused to the mitochondrial targeting sequence of rat liver dimethylglycine dehydrogenase. Translation of 6-HDNO in FAD-supplemented reticulocyte lysate resulted in a protein that contained covalently incorporated FAD, exhibited enzyme activity, and was trypsin-resistant, a characteristic of the tight conformation of the holoenzyme. The attached mitochondrial presequence did not prevent folding, binding of FAD, or enzyme activity of the 6-HDNO moiety of the fusion protein (pre-6-HDNO). Pre-6-HDNO was imported into rat liver mitochondria and processed by the mitochondrial processing peptidase. Incubation of the trypsin-resistant pre-holo-6-HDNO protein with deenergized rat liver mitochondria demonstrated that upon contact with mitochondria, the protein was unfolded and became trypsin sensitive. Mitochondrial import assays showed that the unfolded pre-holo-6-HDNO with covalently attached FAD was imported into rat liver mitochondria. Inside the mitochondrion the holo-6-HDNO was refolded into the trypsin-resistant conformation. However, when pre-apo-6-HDNO was imported only part of the protein became trypsin resistant (approximately 20%). Addition of FAD and the allosteric effector glycerol 3-phosphate to apo-6-HDNO containing mitochondrial matrix was required to transform the protein into the trypsin-resistant conformation characteristic of holo-6-HDNO.
Stoltz
M
M
Biochemisches Institut, Universität Freiburg, Federal Republic of Germany.
Rysavy
P
P
Kalousek
F
F
Brandsch
R
R
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D050893:Dimethylglycine Dehydrogenase; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005415:Flavins / Q000378:metabolism*; D006868:Hydrolysis; D008930:Mitochondria, Liver / Q000201:enzymology*; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D010089:Oxidoreductases, N-Demethylating / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis; D017510:Protein Folding; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014357:Trypsin / Q000494:pharmacology
D005415:Flavins; D024101:Mitochondrial Proteins; D011993:Recombinant Fusion Proteins; D005182:Flavin-Adenine Dinucleotide; D010088:Oxidoreductases; D010089:Oxidoreductases, N-Demethylating; C021413:6-hydroxy-D-nicotine oxidase; D050893:Dimethylglycine Dehydrogenase; C494400:Dmgdh protein, rat; D014357:Trypsin