THE SUBSTITUTION OF A SINGLE AMINO-ACID RESIDUE (SER-116-]ASP) ALTERSNADP-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-ne gative 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 s ubstrates, A novel consensus motif (AGKHVXCEKP) for a class of dehydro genases 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 s hown to have lost tight cofactor binding based on (a) altered tryptoph an fluorescence; (b) lack of NADP liberation through perchloric acid t reatment of the protein; and (c) lack of GFOR enzyme activity, The S11 6D mutant showed glucose dehydrogenase activity (3.6 +/- 0.1 units/mg of protein) with both NADP and NAD as coenzymes (K-m for NADP, 153 +/- 9 mu M; for NAD, 375 +/- 32 mu M. The single site mutation therefore altered GFOR, which in the wild-type situation contains NADP as nondis sociable redox cofactor reacting in a ping-pong type mechanism, to a d ehydrogenase with dissociable NAD(P) as cosubstrate and a sequential r eaction type, After prolonged preincubation of the S116D mutant protei n with excess NADP (but not NAD), GFOR activity could be restored to 7 0 units/mg, one-third of wild-type activity, whereas glucose dehydroge nase activity decreased sharply, A second site mutant (S116D/K121A/K12 3Q/I124K) showed no GFOR activity even after preincubation with NADP, but it retained glucose dehydrogenase activity (4.2 +/- 0.2 units/mg o f protein).