COENZYME SPECIFICITY OF 3-ISOPROPYLMALATE DEHYDROGENASE FROM THERMUS-THERMOPHILUS HB8

The co-enzyme specificity of 3-isopropylmalate dehydrogenase from an extreme thermophile, Thermus thermophilus HB8, was changed from NAD to NADP by site-directed mutagenesis. Based on sequence comparison of 3-isopropylmalate dehydrogenases from various organisms with NAD- and NADP-dependent isocitrate dehydrogenases, Ser226, Ser253 and Ile279 of 3-isopropylmalate dehydrogenase were suggested as determining the co-enzyme specificity. These residues were replaced with the corresponding residues of NADP-dependent isocitrate dehydrogenases; Arg, Gly and Tyr respectively. The single-mutated enzymes, S226R and I279Y, enhanced the activities towards NADP similar to 10- and similar to 3-fold respectively, whereas S253G reduced the activity. Among the multiple-mutated enzymes, the double-mutated S226R/I279Y increased the catalytic efficiency against NADP (similar to 5-fold) and shifted the specificity for NAD towards NADP most significantly (similar to 173-fold).