Expression of the Zymomonas mobilis gfo gene or 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 peptide of 52 amino acid residues. Expression of the gfo gene in cells of Escherichia coli K12, under the control of a tac promoter, led to immunologically detectable proteins in western blots, and to the formation of an enzymatically active precursor form (preGFOR), located in the cytosol. 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 were unstable and rapidly degraded in the periplasm unless an E. coli mutant 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 precursor forms of the respective preGFOR were not degraded. A major protease-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 conformation and that its absence from the E. coli periplasm is responsible for failure to form a stable periplasmic protein. The results strongly suggest that, in Z. mobilis, additional protein factors are required for the transport of NADP across the plasma membrane and/or incorporation of NADP into the GFOR apoenzyme.
Wiegert
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Institut für Biotechnologie 1, Forschungszentrum Jülich GmbH, Germany.
Sahm
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H
Sprenger
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GA
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D010450:Endopeptidases; D004792:Enzyme Precursors / Q000096:biosynthesis* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005798:Genes, Bacterial; D005822:Genetic Vectors; D006868:Hydrolysis; D009249:NADP / Q000494:pharmacology; D010088:Oxidoreductases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000378:metabolism; D013104:Spheroplasts / Q000201:enzymology / Q000378:metabolism; D017355:Zymomonas / Q000201:enzymology* / Q000235:genetics*
D001426:Bacterial Proteins; D004792:Enzyme Precursors; D021382:Protein Sorting Signals; D009249:NADP; D010088:Oxidoreductases; C074291:glucose-fructose oxidoreductase; D010450:Endopeptidases