Efficient flavinylation of glycosomal fumarate reductase by its own ApbE domain in Trypanosoma brucei

Abstract

A subset of flavoproteins has a covalently attached flavin prosthetic group enzymatically attached via phosphoester bonding. In prokaryotes, this is catalysed by ApbE flavin transferases. ApbE-like domains are present in few eukaryotic taxa, e.g. 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 dead ApbE domains. In addition to the trans-flavinylation activity, the ApbE domain catalyses the intramolecular cis-flavinylation with at least 5-fold 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.

Kinetoplastid-specific X2-family kinesins interact with a kinesin-like pleckstrin homology domain protein that localizes to the trypanosomal microtubule quartet

“…Third and finally, a putative flavin-trafficking protein was also found to associate with TbPH1 and TOEFAZ1/CIF1 [28]. This annotation is based on the presence of an ApbE-like domain, which is common in prokaryotes but a rare occurrence in eukaryotic proteins [60]. What an enzyme potentially influencing redox is doing in association with the MtQ is another open question that has been uncovered in this study.…”

“…Also, two proteins were found to be associated with the flagellar pocket, including FP45 (Gheiratmand et al, 2013). The other protein is interestingly a putative flavin-trafficking protein, containing an ApbE-like domain, which is common in prokaryotes but a rare occurrence in eukaryotic proteins (Schenk et al, 2021). What an enzyme potentially influencing redox is doing in association with the MtQ is another open question that has been uncovered in this study.…”