Co-enzyme specificity of 3-isopropylmalate dehydrogenase from Thermits thermophilus HB8

The co–enzyme specificity of 3–isopropylmalate dehydrogenasefrom an extreme thermophile, Thermus thermophilus HB8, was changedfrom NAD to NADP by site–directed mutagenesis Based onsequence comparison of 3–isopropylmalate dehydrogenasesfrom various organisms with NAD– and NADP–dependentisocitrate dehydrogenases, Ser226, Ser253 and De279 of 3–isopropylmalatedehydrogenase were suggested as determining the co–enzymespecificity. These residues were replaced with the correspondingresidues of NADP–dependent isocitrate dehydrogenases;Arg, Gly and Tyr respectively. The single–mutated enzymes,S226R and I279Y, enhanced the activities towards NADP 10–and 3–fold respectively, whereas S253G reduced the activity.Among the multiple–mutated enzymes, the double–mutatedS226R/I279Y increased the catalytic efficiency against NADP( fold) and shifted the specificity for NAD towards NADP mostsignificantly ( 173–fold).