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.
Institut für Biotechnologie 1, Jülich, Germany Institut für Genetik, Universität Bayreuth, Bayreuth, Germany.
D
D
Halbig
T
T
Wiegert
N
N
Blaudeck
R
R
Freudl
G A
GA
Sprenger
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