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The enzymes responsible for sorbitol formation in Zymomonas mobiis were investigated. A previously undescribed enzyme catalyzes the intermolecular oxidation-reduction of glucose and fructose to form gluconolactone and sorbitol. This enzyme has been purified; it had a subunit size of 40,000 daltons and is probably tetrameric at low pH. It contained tightly bound NADP as the hydrogen carrier and did not require any added cofactor for activity. In addition, a gluconolactonase has been isolated, although not completely purified. Together these two enzymes were capable of completely converting a 54% (wt/vol) equimolar mixture of glucose and fructose to sorbitol and sodium gluconate at the optimum pH of close to 6.2. The oxidoreductase had low affinities for its substrates, but natural environmental conditions would expose it to high concentrations of sugars. The amount of the enzyme in Z. mobilis cells was sufficient to account for the rate of sorbitol formation in vivo. However, the enzyme was present in the highest amounts when the cells were grown on glucose alone, and it was repressed by the presence of fructose; this was not the case with the gluconolactonase.In 1984 it was noted that among the fermentation products of the ethanol producer Zymomonas mobilis, sorbitol accumulated to quite high levels when sucrose or a mixture of glucose and fructose was used as the carbon source (5). The carbon skeleton of sorbitol was shown to derive exclusively from fructose. Several possible enzymatic routes for sorbitol production have been suggested (23, 24); however, the levels of the proposed enzymes in extracts of Z. mobilis were far too low to account for the rate of sorbitol production in vivo. We proposed (16) that two dehydrogenases acting through an unknown bound cofactor were responsible for the dehydrogenation of glucose to gluconolactone, followed by the reduction of fructose to sorbitol. Although the activity of a glucose dehydrogenase measured with an artificial acceptor such as ferricyanide or dichlorophenolindophenol was equivalent to the required activity, there remained doubts about its role in sorbitol production because of the differing sugar specificities of the two processes. Nevertheless, an extract of cells, even after removal of endogenous salts and cofactors, was capable of reaction with glucose and fructose (and no other combination of sugars) to produce both sorbitol and gluconic acid without any added cofactor.In this paper we show that sorbitol and gluconolactone production from fructose and glucose in Z. mobilis is catalyzed by a single enzyme containing tightly bound NADP. In addition, there is a gluconolactonase (EC 3.1.1.17) which accelerates hydrolysis of the unstable lactone. The new enzyme has provisionally been named D-glucose-l:D-fructose-2-oxidoreductase. It has been purified, and information on its structural and kinetic properties was obtained. In the same purification procedure, gluconolactonase has also been isolated, although not fully purified.The glucose-fructose oxidoreductase t...

1. The two alcohol dehydrogenases found in Zymomonas mobilis have each been purified using dye-ligand chromatography and a f h i t y elution with nucleotides.2. The isoenzyme with lower electrophoretic mobility (ZADH-1) is a zinc enzyme with properties essentially similar to preparations described elsewhere.3. The faster isoenzyme (ZADH-2) accounted for some 90% of the ethanol-oxidizing activity in freshly prepared extracts and corresponded to the iron-activated enzyme previously described. This enzyme was inactivated by zinc; activity could only be retained during purification by including either ferrous ions or cobaltous ions in the buffers.4.ZADH-2 has relatively low acetaldehyde reductase activity; consequently ZADH-1 is responsible for about half of the physiological activity (acetaldehyde reduction) in Zymomonas cells.5. Kinetic studies showed that ZADH-2 is activated by ethanol in both reaction directions; a hypothesis for the mechanism of activation is presented.6 . Metal ion analyses of ZADH-2 prepared in the presence of iron or cobalt indicated one atom of the relevant metal per subunit, with no significant zinc content.7. N-terminal sequence analyses showed that the ZADH-1 has some homology with the Bacillus stearothermophilus enzyme, whereas ZADH-2 resembles the yeast enzyme more closely.Alcohol dehydrogenases from the bacterium Zymomonas mobilis have been isolated by at least four groups [l-41; however there seems to be some disagreement concerning the properties of the purified enzymes. Since the sources of the bacterial cells were different and they were grown in different conditions in each case, it is possible that genetically distinct alcohol dehydrogenases have been described. Two bands of activity staining for alcohol dehydrogenase after electrophoresis were reported [l, 31 and each separately isolated. The main (slower) components had high specific activity and were tetramers. The faster components in each case had very low specific activities, were dimers and were considered to be degradation products or artefacts of the extraction method.We described an alcohol dehydrogenase isolated from Z . mobilis [4] having a high specific activity which could only be maintained in the presence of ferrous ions. Complexing agents inactivated the purified enzyme and the activity could be fully restored only with ferrous ions; cobaltous ions restored up to half the activity, but other divalent metal ions, including zinc, were ineffective. This contrasts with the earlier report [l] that each of the two Zymomonas isoenzymes contains zinc (like most other alcohol dehydrogenases); we could find only trace amounts of zinc in our iron-activated enzyme.Correspondence to R. K . Scopes, Department of Biochemistry, La Trobe University, Bundoora, Victoria, Australia 3083Abbreviutions. ZADH-1, Zymomonas mobilis alcohol dehydrogenase isoenzyme-1 ; ZADH-2, Zymomonas mobilis alcohol dehydrogenase isoenzyme-2; SDS, sodium dodecyl sulphate.Enzyme. Alcohol dehydrogenase (EC 1.1.1.1).The present paper describes a simple on...

In GFOR, the NADP is found associated with a classical dinucleotide-binding domain in a conventional fashion. The NADP is effectively buried in the protein-subunit interior as a result of interactions with the N-terminal arm from an adjacent subunit in the tetramer, and with a short helix from the C-terminal domain of the protein. This accounts for NADP's inability to dissociate. The N-terminal arm may also contribute to stabilization of the tetramer. The enzyme has an unexpected structural similarity with the cytoplasmic enzyme glucose-6-phosphate dehydrogenase (G6PD). We hypothesize that both enzymes have diverged from a common ancestor. The mechanism of catalysis is still unclear, but we have identified a conserved structural motif (Glu-Lys-Pro) in the active site of GFOR and G6PD that may be important for catalysis.

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