EXPORT OF THE PERIPLASMIC NADP-CONTAINING GLUCOSE-FRUCTOSE OXIDOREDUCTASE OF ZYMOMONAS-MOBILIS

Glucose-fructose oxidoreductase (GFOR) of the gram-negative bacterium Zymomonas mobilis is a periplasmic enzyme with the tightly bound cofac tor NADP. The preprotein carries an unusually long N-terminal signal s equence of 52 amino acid residues. A sorbitol-negative mutant strain ( ACM3963) was found toe deficient in GFOR activity and was used for the expression of plasmid-borne copies of the wild-type gfo gene or of al leles encoding alterations in the signal sequence of the pre-GFOR prot ein. Z. mobilis cells with the wild-type gfo allele translocated pre-G FOR, at least partially, via the Sec pathway since CCCP (carboxylcyani de-m-chlorophenyl- hydrazone: uncoupler of proton motive force) or sod ium azide (inhibitor of SecA) abolished the processing of GFOR. A gfo allele with the hydrophobic region of the signal sequence removed (res idues 32-46; Delta 32-46) led to a protein that was no longer processe d, but showed full enzymatic activity (180 U/mg) and had the cofactor NADP firmly bound. A deletion in the n-region of the signal sequence ( residues 2-20; Delta 2-20) Or exchange of tire entire GFOR signal sequ ence with the signal sequence of gluconolactonase of Z. mobilis led to active and processed GFOR. Strain ACM3963 could not grow in the prese nce of high sugar concentrations (1 M sucrose) unless sorbitol was add ed. The presence of the plasmid-borne gfo wild-type allele or of the D elta 2-20 deletion led to the restoration of growth on media with 1 M sucrose, whereas the presence of the Delta 32-46 deletion led to a gro wth behavior similar to that of strain ACM3963, with no sorbitol forma tion from sucrose.