title: Detrimental effect of the 6 His C-terminal tag on YedY enzymatic activity and influence of the TAT signal sequence on YedY synthesis.
abstract: BACKGROUND
  YedY, a molybdoenzyme belonging to the sulfite oxidase family, is found in most Gram-negative bacteria. It contains a twin-arginine signal sequence that is cleaved after its translocation into the periplasm. Despite a weak reductase activity with substrates such as dimethyl sulfoxide or trimethylamine N-oxide, its natural substrate and its role in the cell remain unknown. Although sequence conservation of the YedY family displays a strictly conserved hydrophobic C-terminal residue, all known studies on Escherichia coli YedY have been performed with an enzyme containing a 6 histidine-tag at the C-terminus which could hamper enzyme activity.
  
  
  RESULTS
  In this study, we demonstrate that the tag fused to the C-terminus of Rhodobacter sphaeroides YedY is detrimental to the enzyme's reductase activity and results in an eight-fold decrease in catalytic efficiency. Nonetheless this C-terminal tag does not influence the properties of the molybdenum active site, as assayed by EPR spectroscopy. When a cleavable His-tag was fused to the N-terminus of the mature enzyme in the absence of the signal sequence, YedY was expressed and folded with its cofactor. However, when the signal sequence was added upstream of the N-ter tag, the amount of enzyme produced was approximately ten-fold higher.
  
  
  CONCLUSIONS
  Our study thus underscores the risk of using a C-terminus tagged enzyme while studying YedY, and presents an alternative strategy to express signal sequence-containing enzymes with an N-terminal tag. It brings new insights into molybdoenzyme maturation in R. sphaeroides showing that for some enzymes, maturation can occur in the absence of the signal sequence but that its presence is required for high expression of active enzyme.
authors.affiliation: CEA, IBEB, Laboratoire de Bioénergétique Cellulaire, Saint-Paul-lez-Durance F-13108, France. monique.sabaty@cea.fr.
authors.forename: Monique
authors.initials: M
authors.lastname: Sabaty
authors.forename: Sandrine
authors.initials: S
authors.lastname: Grosse
authors.forename: Geraldine
authors.initials: G
authors.lastname: Adryanczyk
authors.forename: Séverine
authors.initials: S
authors.lastname: Boiry
authors.forename: Frédéric
authors.initials: F
authors.lastname: Biaso
authors.forename: Pascal
authors.initials: P
authors.lastname: Arnoux
authors.forename: David
authors.initials: D
authors.lastname: Pignol
mesh_terms: D020134:Catalytic Domain; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008982:Molybdenum / Q000737:chemistry; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism*; D010088:Oxidoreductases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012242:Rhodobacter sphaeroides / Q000201:enzymology*
chemical_list: C471213:His-His-His-His-His-His; D009842:Oligopeptides; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D006639:Histidine; D008982:Molybdenum; D010088:Oxidoreductases