EXPRESSION OF THE ZYMOMONAS-MOBILIS GFO GENE FOR NADP-CONTAINING GLUCOSE-FRUCTOSE OXIDOREDUCTASE (GFOR) IN ESCHERICHIA-COLI - FORMATION OF ENZYMATICALLY ACTIVE PREGFOR BUT LACK OF PROCESSING INTO A STABLE PERIPLASMIC PROTEIN

Glucose:fructose oxidoreductase (GFOR) of the gram-negative bacterium Zymomonas mobilis is a periplasmic enzyme with tightly bound cofactor NADP. The preprotein carries an unusually long N-terminal signal pepti de of 52 amino acid residues. Expression of the gfo gene in cells of E scherichia coli K12, under the control of a tac promoter, led to immun ologically detectable proteins in western blots, and to the formation of an enzymatically active precursor form (preGFOR), located in the cy tosol. Processing of preGFOR to the mature form was not observed in E. coli. Replacement of the authentic GFOR signal peptide by the shorter signal peptides of PhoA or OmpA from E. coli led to processing of the respective GFOR precursor proteins. However, the processed proteins w ere unstable and rapidly degraded in the periplasm unless an E. call m utant was used that carried a triple lesion for periplasmic and outer- membrane proteases. When fusion-protein export was inhibited by sodium azide or carboxylcyanide m-chlorophenylhydrazone, the cytoplasmic pre cursor forms of the respective preGFOR were not degraded. A major prot ease-resistant GFOR peptide from the OmpA-GFOR fusion was found within spheroplasts of E. coli to which NADP had been added externally. The formation of this peptide did not occur in the presence of NAD. It is concluded that NADP is required for GFOR to fold into its native confo rmation and that its absence from the E. coli periplasm is responsible for failure to form a stable periplasmic protein. The results strongl y suggest that, in Z. mobilis, additional protein factors are required for the transport of NADP across the plasma membrane and/or incorpora tion of NADP into the GFOR apoenzyme.