Folding, Flavinylation, and Mitochondrial Import of 6-Hydroxy-D-nicotine Oxidase Fused to the Presequence of Rat Dimethylglycine Dehydrogenase

Abstract

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.

Covalent attachment of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) to enzymes: The current state of affairs

“…Recent studies have focused on the requirement for flavinylation in the mitochondrial import of 6HDNO-dimethylglycine dehydrogenase fusions (Stoltz et al, 1995), the interaction of 6HDNO with the chaperone protein GroE (Brandsch et al, 1992), and the covalent incorporation of flavin analogues (modified in the adenine moiety of FAD) into the active site of the enzyme (Stoltz et al, 1996a(Stoltz et al, , 1996b. In oxidase, where the FAD-linking His residue was replaced with Cys, noncovalently bound FAD could be displaced rapidly by added 8-methylsulfonyl-FAD or 8-chloro-FAD (Stolz et al, 1996b).…”

Molecular cloning and tissue distribution of rat sarcosine dehydrogenase

“…Isolation of rat liver mitochondria was performed according to the method of Conboy et al [21] and fractionation into matrix and membrane subfractions were performed by freezing in liquid nitrogen and thawing the mitochondria (four times) followed by centrifugation at 100 000 g at 2°C for 30 min as previously described [22].…”

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

“…The boxed sequence corresponds to peptides introduced by the cloning procedure. The arrow indicates the MPP cleavage site, according to [29]. Bold style indicates the histidine residue that covalently binds FAD.…”