The efficient export of NADP-containing glucose-fructose oxidoreductase tothe periplasm of Zymomonas mobilis depends both on an intact twin-argininemotif 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 gra m-negative bacterium Zymomonas mobilis belongs to a class of redox cofactor -dependent enzymes which are exported with the aid of a signal peptide cont aining a so-called twin-arginine motif. In this paper we show that the repl acement of one or both arginine residues results in drastically reduced tra nslocation of glucose-fructose oxidoreductase to the periplasm, showing tha t this motif is essential. Mutant proteins which, in contrast to wild-type glucose-fructose oxidoreductase, bind NADP in a looser and dissociable mann er, 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 r ecognizes, as an additional signal, a conformational change in the structur e of the protein, most likely triggered by cofactor binding. Furthermore, t hese results suggest that glucose-fructose oxidoreductase is exported in a folded form. A glucose-fructose oxidoreductase:beta-galactosidase fusion pr otein is not lethal to Z. mobilis cells and leads to the accumulation of th e cytosolic preform of wild-type glucose-fructose oxidoreductase expressed in trans but not of a typical Sec-substrate (OmpA), indicating that the glu cose-fructose oxidoreductase translocation apparatus can be blocked without interfering with the export of essential proteins via the Sec pathway.