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 Available to Purchase

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 pH10.0 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(²H) 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-Xaa₄-Asn-Xaa₂-His (where ‘Xaa’ denotes ‘any amino acid').