The substitution of a single amino acid residue (Ser-116 --> Asp) alters NADP-containing glucose-fructose oxidoreductase of Zymomonas mobilis into a glucose dehydrogenase with dual coenzyme specificity.
Glucose-fructose oxidoreductase (GFOR, EC 1.1.1.99.-) from the Gram-negative bacterium Zymomonas mobilis contains the tightly bound cofactor NADP. Based on the revision of the gfo DNA sequence, the derived GFOR sequence was aligned with enzymes catalyzing reactions with similar substrates. A novel consensus motif (AGKHVXCEKP) for a class of dehydrogenases was detected. From secondary structure analysis the serine-116 residue of GFOR was predicted as part of a Rossmann-type dinucleotide binding fold. An engineered mutant protein (S116D) was purified and shown to have lost tight cofactor binding based on (a) altered tryptophan fluorescence; (b) lack of NADP liberation through perchloric acid treatment of the protein; and (c) lack of GFOR enzyme activity. The S116D mutant showed glucose dehydrogenase activity (3.6 +/- 0.1 units/mg of protein) with both NADP and NAD as coenzymes (Km for NADP, 153 +/- 9 microM; for NAD, 375 +/- 32 microM). The single site mutation therefore altered GFOR, which in the wild-type situation contains NADP as nondissociable redox cofactor reacting in a ping-pong type mechanism, to a dehydrogenase with dissociable NAD(P) as cosubstrate and a sequential reaction type. After prolonged preincubation of the S116D mutant protein with excess NADP (but not NAD), GFOR activity could be restored to 70 units/mg, one-third of wild-type activity, whereas glucose dehydrogenase activity decreased sharply. A second site mutant (S116D/K121A/K123Q/I124K) showed no GFOR activity even after preincubation with NADP, but it retained glucose dehydrogenase activity (4.2 +/- 0.2 units/mg of protein).
Wiegert
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Institut für Biotechnologie 1 der Forschungszentrum Jülich GmbH, D-52425 Jülich, Germany.
Sahm
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Sprenger
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D000595:Amino Acid Sequence; D001216:Asparagine / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003067:Coenzymes / Q000235:genetics / Q000378:metabolism; D042843:Glucose 1-Dehydrogenase; D005948:Glucose Dehydrogenases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009249:NADP / Q000235:genetics / Q000378:metabolism; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D017354:Point Mutation; D016415:Sequence Alignment; D012694:Serine / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics; D017355:Zymomonas / Q000201:enzymology* / Q000235:genetics
D003067:Coenzymes; D012694:Serine; D009249:NADP; D001216:Asparagine; D010088:Oxidoreductases; D005948:Glucose Dehydrogenases; D042843:Glucose 1-Dehydrogenase; C074291:glucose-fructose oxidoreductase