The Conserved RGxxE Motif of the Bacterial FAD Assembly Factor SdhE Is Required for Succinate Dehydrogenase Flavinylation and Activity

Abstract

Succinate dehydrogenase (SDH) is an important respiratory enzyme that plays a critical role in the generation of energy in the majority of eukaryotes, bacteria, and archaea. The activity of SDH is dependent on the covalent attachment of the redox cofactor FAD to the flavoprotein subunit SdhA. In the Gram-negative bacteria Escherichia coli and Serratia sp. ATCC 39006, the covalent attachment of FAD to SdhA is dependent on the FAD assembly factor SdhE (YgfY). Although mechanisms have been proposed, experimental evidence that elucidates the molecular details of SdhE-mediated flavinylation are scarce. In this study, truncation and alanine swap mutagenesis of SdhE identified a highly conserved RGxxE motif that was important for SdhE function. Interestingly, RGxxE site-directed variants were not impaired in terms of protein folding or interactions with SdhA. Purification and analysis of SdhA from different mutant backgrounds demonstrated that SdhE interacts with and flavinylates folded SdhA without a requirement for the assembly of the entire SDH complex. SdhA was also partially active in the absence of SdhE, suggesting that SdhA is able to attach FAD through an inefficient autocatalytic mechanism. The results presented are of widespread relevance because SdhE and SDH are required for bacterial pathogenesis and mutations in the eukaryotic homologues of SdhE and SDH are associated with cancer in humans.

The succinate dehydrogenase assembly factor, SdhE, is required for the flavinylation and activation of fumarate reductase in bacteria

“…These results suggest that Glu19 is important for SDH, but not FRD, activation. The SdhE(G16R) variant cannot flavinylate or activate SDH [16] or FRD, yet was not impaired for interaction with FrdA. Combined, these results suggest that the RGxxE motif of SdhE homologues is important for the flavinylation of both SDH and FRD.…”

“…SdhE variants of the RGxxE motif are impaired for flavinylation and activation of SDH [16]. To investigate if SdhE flavinylated FRD with a similar mechanism, seven Serratia 39006 SdhE site‐directed variants were assessed in phenotypic rescue assays of E. coli Δ sdhE ::Kan mutants.…”

“…The G16R and E19A SdhE variants are unable to fully flavinylate or activate SDH, yet still interact with SdhA [16]. To investigate if the SdhE variants (R15A) and (G16R) were impaired for interactions with FrdA, co‐immunoprecipitation experiments were performed using His‐FrdA (Bait) and SdhE(WT, R15A, G16R)‐FLAG (Prey) on an anti‐FLAG resin.…”

Redox State of Flavin Adenine Dinucleotide Drives Substrate Binding and Product Release in Escherichia coli Succinate Dehydrogenase

“…How is the FAD cofactor recruited to the apoenzyme and how does flavinylation proceed thereafter? In the case of SdhCDAB, it was recently discovered that the SdhE chaperone directly interacts with the SdhA subunit to mediate flavinylation (32, 48), and the absence of the equivalent SdhAF2 chaperone in humans results in paraganglioma tumorigenesis (33). Following insertion of FAD into its binding pocket and repositioning of the capping domain, covalent attachment is thought to be an autocatalytic process, a common theme that is conserved in other flavoenzymes (49–51).…”

Draft Genome Sequence of Serratia sp. Strain ATCC 39006, a Model Bacterium for Analysis of the Biosynthesis and Regulation of Prodigiosin, a Carbapenem, and Gas Vesicles

“…For example, SdhE was recently investigated in this strain. SdhE is widely conserved in eukaryotes and Alpha -, Beta -, and Gammaproteobacteria and is essential for flavinylation and activation of succinate dehydrogenase, an enzyme central to the electron transport chain and the tricarboxylic acid cycle (17, 19, 20). …”