The conserved RGxxE motif of the bacterial FAD assembly factor SdhE is required for succinate dehydrogenase flavinylation and activity.
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.
Department of Microbiology and Immunology, University of Otago , P.O. Box 56, Dunedin 9054, New Zealand.
Matthew B
MB
McNeil
Peter C
PC
Fineran
D020816:Amino Acid Motifs; D001059:Apoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017124:Conserved Sequence; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry* / Q000378:metabolism; D005420:Flavoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020035:Holoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D055550:Protein Stability; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012705:Serratia / Q000201:enzymology* / Q000378:metabolism; D013385:Succinate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D001059:Apoproteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D005420:Flavoproteins; D020035:Holoenzymes; D007527:Isoenzymes; D050505:Mutant Proteins; D010446:Peptide Fragments; D021122:Protein Subunits; D011994:Recombinant Proteins; C084271:SdhA protein, Bacteria; D014157:Transcription Factors; C510287:YgfY protein, E coli; D005182:Flavin-Adenine Dinucleotide; D013385:Succinate Dehydrogenase