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 kcat 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').
Abbreviations used: ADH, alcohol dehydrogenase; KIE, primary 2H kinetic isotope effect; K295A, etc., mutated enzyme bearing a Lys-295→Ala replacement, etc.; M2DH, d-mannitol 2-dehydrogenase; MDR, zinc-dependent medium-chain dehydrogenase/reductase; NADD, 4-S-4-[2H]NADH; PSLDR, polyol-specific long-chain dehydrogenase/reductase; (Ps)M2DH, M2DH (from Pseudomonas fluorescens); SDR, short-chain dehydrogenase/reductase; S-KIE, solvent 2H kinetic isotope effect.