The efficient export of NADP-containing glucose-fructose oxidoreductase to the periplasm of Zymomonas mobilis depends both on an intact twin-arginine motif in the signal peptide and on the generation of a structural export signal induced by cofactor binding.
The periplasmic, NADP-containing glucose-fructose oxidoreductase of the gram-negative bacterium Zymomonas mobilis belongs to a class of redox cofactor-dependent enzymes which are exported with the aid of a signal peptide containing a so-called twin-arginine motif. In this paper we show that the replacement of one or both arginine residues results in drastically reduced translocation of glucose-fructose oxidoreductase to the periplasm, showing that this motif is essential. Mutant proteins which, in contrast to wild-type glucose-fructose oxidoreductase, bind NADP in a looser and dissociable manner, were severely affected in the kinetics of plasma membrane translocation. These results strongly suggest that the translocation of glucose-fructose oxidoreductase into the periplasm uses a Sec-independent apparatus which recognizes, as an additional signal, a conformational change in the structure of the protein, most likely triggered by cofactor binding. Furthermore, these results suggest that glucose-fructose oxidoreductase is exported in a folded form. A glucose-fructose oxidoreductase:beta-galactosidase fusion protein is not lethal to Z. mobilis cells and leads to the accumulation of the cytosolic preform of wild-type glucose-fructose oxidoreductase expressed in trans but not of a typical Sec-substrate (OmpA), indicating that the glucose-fructose oxidoreductase translocation apparatus can be blocked without interfering with the export of essential proteins via the Sec pathway.
Halbig
D
D
Institut für Biotechnologie 1, Jülich, Germany Institut für Genetik, Universität Bayreuth, Bayreuth, Germany.
Wiegert
T
T
Blaudeck
N
N
Freudl
R
R
Sprenger
G A
GA
D001120:Arginine / Q000502:physiology*; D004926:Escherichia coli / Q000201:enzymology; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D019897:Periplasm / Q000378:metabolism*; D011485:Protein Binding; D017510:Protein Folding; D011993:Recombinant Fusion Proteins; D013997:Time Factors; D017355:Zymomonas / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D009249:NADP; D001120:Arginine; D010088:Oxidoreductases; C074291:glucose-fructose oxidoreductase