Co-enzyme specificity of 3-isopropylmalate dehydrogenase from Thermits thermophilus HB8
- 1 March 1994
- journal article
- research article
- Published by Oxford University Press (OUP) in Protein Engineering, Design and Selection
- Vol. 7 (3) , 401-403
- https://doi.org/10.1093/protein/7.3.401
Abstract
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 De279 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 ∼10– and ∼3–fold respectively, whereas S253G reduced the activity. Among the multiple–mutated enzymes, the double–mutated S226R/I279Y increased the catalytic efficiency against NADP ( ∼ fold) and shifted the specificity for NAD towards NADP most significantly (∼ 173–fold).Keywords
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