Efficient flavinylation of glycosomal fumarate reductase by its own ApbE domain in Trypanosoma brucei.
A subset of flavoproteins has a covalently attached flavin prosthetic group enzymatically attached via phosphoester bonding. In prokaryotes, this is catalysed by alternative pyrimidine biosynthesis E (ApbE) flavin transferases. ApbE-like domains are present in few eukaryotic taxa, for example the N-terminal domain of fumarate reductase (FRD) of Trypanosoma, a parasitic protist known as a tropical pathogen causing African sleeping sickness. We use the versatile reverse genetic tools available for Trypanosoma to investigate the flavinylation of glycosomal FRD (FRDg) in vivo in the physiological and organellar context. Using direct in-gel fluorescence detection of covalently attached flavin as proxy for activity, we show that the ApbE-like domain of FRDg has flavin transferase activity in vivo. The ApbE domain is preceded by a consensus flavinylation target motif at the extreme N terminus of FRDg, and serine 9 in this motif is essential as flavin acceptor. The preferred mode of flavinylation in the glycosome was addressed by stoichiometric expression and comparison of native and catalytically inactive ApbE domains. In addition to the trans-flavinylation activity, the ApbE domain catalyses the intramolecular cis-flavinylation with at least fivefold higher efficiency. We discuss how the higher efficiency due to unusual fusion of the ApbE domain to its substrate protein FRD may provide a selective advantage by faster FRD biogenesis during rapid metabolic adaptation of trypanosomes. The first 37 amino acids of FRDg, including the consensus motif, are sufficient as flavinylation target upon fusion to other proteins. We propose FRDg(1-37) as 4-kDa heat-stable, detergent-resistant fluorescent protein tag and suggest its use as a new tool to study glycosomal protein import.
['Schenk|Robin|R|0000-0001-6823-8128', 'Bachmaier|Sabine|S|0000-0001-7416-8613', 'Bringaud|Frédéric|F|0000-0003-4552-6877', 'Boshart|Michael|M|0000-0002-5070-2663']
D004138:Dinitrocresols D005420:Flavoproteins D006801:Humans D000072417:Protein Domains D021381:Protein Transport D011743:Pyrimidines D013385:Succinate Dehydrogenase D014166:Transferases D014346:Trypanosoma brucei brucei D014353:Trypanosomiasis, African D014364:Tryptophan
glycosome fumarate reductase ApbE flavin transferase fluorescent tag