A catalytic consensus motif for D-mannitol 2-dehydrogenase, a member of a polyol-specific long-chain dehydrogenase family, revealed by kinetic characterization of site-directed mutants of the enzyme from Pseudomonas fluorescens.
Lys-295, Asn-300 and His-303 of D-mannitol 2-dehydrogenase from Pseudomonas fluorescens were mutated individually into alanine (K295A, N300A and H303A respectively). Purified mutants displayed catalytic efficiencies for NAD(+)-dependent oxidation of D-mannitol 300-fold (H303A), 1000-fold (N300A) and approx. 400000-fold (K295A) below the wild-type level. Comparison of primary kinetic isotope effects on kinetic parameters for D-fructose reduction by wild-type and mutants at pH 10.0 demonstrate that Asn-300 has an auxiliary role in stabilization of the transition state of hydride transfer, and His-303 contributes to substrate positioning. The large solvent isotope effect of 11+/-1 on k (cat) for mannitol oxidation by K295A at pH((2)H) 10.5 suggests a role for Lys-295 in general base enzymic catalysis. Positional conservation of Lys-295, Asn-300 and His-303 across a family of polyol-specific long-chain dehydrogenases suggests a unique catalytic signature: Lys-Xaa(4)-Asn-Xaa(2)-His (where 'Xaa' denotes 'any amino acid').
Klimacek
Mario
M
Institute of Biotechnology, Graz University of Technology, Petersgasse 12/I, A-8010 Graz, Austria.
Nidetzky
Bernd
B
D000426:Alcohol Dehydrogenase / Q000737:chemistry; D000429:Alcohol Oxidoreductases / Q000737:chemistry*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry; D020134:Catalytic Domain; D017124:Conserved Sequence; D006863:Hydrogen-Ion Concentration; D007554:Isotopes; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008353:Mannitol / Q000494:pharmacology; D008354:Mannitol Dehydrogenases; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017434:Protein Structure, Tertiary; D011551:Pseudomonas fluorescens / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D007554:Isotopes; D011994:Recombinant Proteins; D008353:Mannitol; D001216:Asparagine; D000429:Alcohol Oxidoreductases; D008354:Mannitol Dehydrogenases; D000426:Alcohol Dehydrogenase; C116789:mannitol 2-dehydrogenase (NADP); D008239:Lysine