Characterization and multiple molecular forms of TorD from Shewanella massilia, the putative chaperone of the molybdoenzyme TorA.
Several bacteria use trimethylamine N-oxyde (TMAO) as an exogenous electron acceptor for anaerobic respiration. This metabolic pathway involves expression of the tor operon that codes for a periplasmic molybdopterin-containing reductase of the DMSO/TMAO family, a pentahemic c-type cytochrome, and the TorD cytoplasmic chaperone, possibly required for acquisition of the molybdenum cofactor and translocation of the reductase by the twin-arginine translocation system. In this report, we show that the TorD chaperone from Shewanella massilia forms multiple and stable oligomeric species. The monomeric, dimeric, and trimeric forms were purified to homogeneity and characterized by analytical ultracentrifugation. Small-angle X-ray scattering (SAXS) and preliminary diffraction data indicated that the TorD dimer is made of identical protein modules of similar size to the monomeric species. Interconversion of the native oligomeric forms occurred at acidic pH value. In this condition, ANS fluorescence indicates a non-native conformation of the polypeptide chain in which, according to the circular dichroism spectra, the alpha-helical content is similar to that of the native species. Surface plasmon resonance showed that both the monomeric and dimeric species bind the mature TorA enzyme, but that the dimer binds its target protein more efficiently. The possible biologic significance of these oligomers is discussed in relation to the chaperone activity of TorD, and to the ability of another member of the TorD family to bind the Twin Arginine leader sequences of the precursor of DMSO/TMAO reductases.
Groupe de Cristallographie Biologique, Institut de Pharmacologie et Biologie Structurale, 31077 Toulouse Cedex, France.
Samuel
S
Tranier
Isabelle
I
Mortier-Barrière
Marianne
M
Ilbert
Catherine
C
Birck
Chantal
C
Iobbi-Nivol
Vincent
V
Méjean
Jean-Pierre
JP
Samama
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D019281:Dimerization; D029968:Escherichia coli Proteins; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism*; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020836:Protein Structure, Quaternary; D020592:Shewanella / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D020349:Surface Plasmon Resonance
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; D020033:Protein Isoforms; C088083:TorD protein, E coli; D010089:Oxidoreductases, N-Demethylating; C023186:trimethylamine dehydrogenase