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On the basis of sequence comparison between NAD+-dependent leucine dehydrogenase (LeuDH) from Thermoactinomyces intermedius and NADP+-dependent dehydrogenases, a set of amino acid residues that are supposed to determine the coenzyme specificity of LeuDH were assigned. Systematic replacement of these amino acids by others was done with the aim to switch its natural coenzyme specificity to a new one preferring NADP+. Single D203A, double D203A-I204R and triple D203A-I204R-D210R mutation enzymes were constructed. The wild-type LeuDH is inactive with NADP+. However, D203A single mutant exhibited dual specificity for NAD+ and NADP+ with essentially identical k(cat)/Km values for both coenzymes, but the values were three orders of magnitude lower than that of the wild-type enzyme. Introduction of positive charge at 204 together with the removal of the negative charge at 203 in the double mutant D203A-I204R provided the enzyme with significantly high affinity for NADP+. The best k(cat)/Km value for NADP+ was shown for the triple mutant D203A-I204R-D210R: more than 2% of the k(cat)/Km value of the wild-type enzyme. Thus, we succeeded in constructing a mutant LeuDH with a new coenzyme specificity preferring NADP+ which is highly active (specific activity, 19 micromol/mg/min).

Construction of a new leucine dehydrogenase with preferred specificity for NADP+ by site-directed mutagenesis of the strictly NAD+-specific enzyme