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 cofactor NADP. The preprotein carries an unusually long N-terminal signal sequence of 52 amino acid residues. A sorbitol-negative mutant strain (ACM3963) was found to be deficient in GFOR activity and was used for the expression of plasmid-borne copies of the wild-type gfo gene or of alleles encoding alterations in the signal sequence of the pre-GFOR protein. Z. mobilis cells with the wild-type gfo allele translocated pre-GFOR, at least partially, via the Sec pathway since CCCP (carboxylcyanide-m-chlorophenylhydrazone; uncoupler of proton motive force) or sodium azide (inhibitor of SecA) abolished the processing of GFOR. A gfo allele with the hydrophobic region of the signal sequence removed (residues 32-46; Delta32-46) led to a protein that was no longer processed, 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; Delta2-20) or exchange of the entire GFOR signal sequence with the signal sequence of gluconolactonase of Z. mobilis led to active and processed GFOR. Strain ACM3963 could not grow in the presence of high sugar concentrations (1 M sucrose) unless sorbitol was added. The presence of the plasmid-borne gfo wild-type allele or of the Delta2-20 deletion led to the restoration of growth on media with 1 M sucrose, whereas the presence of the Delta32-46 deletion led to a growth behavior similar to that of strain ACM3963, with no sorbitol formation from sucrose.
Wiegert
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Institut für Biotechnologie 1, Forschungszentrum Jülich GmbH, Postfach 1913, D-52425 Jülich, Germany.
Sahm
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Sprenger
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D001483:Base Sequence; D008969:Molecular Sequence Data; D009154:Mutation; D010088:Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D017355:Zymomonas / Q000201:enzymology* / Q000235:genetics
D010088:Oxidoreductases; C074291:glucose-fructose oxidoreductase