Reversal of the nucleotide specificity of ketol acid reductoisomerase by site-directed mutagenesis identifies the NADPH binding site.
Analysis of the published amino acid sequences of the enzyme ketol acid reductoisomerase (KARI) from seven organisms identified three regions with highly conserved sequences. One of these regions is predicted to be the dinucleotide fold where NADPH binds. In order to confirm that this region did include the NADPH binding site, we used oligonucleotide-mediated site-directed mutagenesis to study the function of specific amino acids in this region in terms of their interactions with NADPH. Four positively charged amino acids, R68, K69, K75, and R76, were mutated singly, in different combinations, and finally as a quartet in order to evaluate electrostatic interactions with NADPH. Mutation of each of the arginines singly to glutamine results in a 60- to 100-fold reduction in k(cat)/K(m) for NADPH. Mutation of each of the lysines singly does not significantly alter the steady state kinetic parameters associated with NADPH. None of these mutations significantly alters the affinity of the enzyme for NADH. After looking at double mutations of these four amino acids, we constructed the quadruplet mutant R68DK69LK75VR76D. This mutant has K(m) and k(cat) values of 19.3 microM and 5.3 min(-1) for NADH, which compares to 207 microM and 0.11 min(-1) for the wild-type enzyme. For the quadruplet mutant the corresponding values for NADPH are >200 microM for K(m) and 2 min(-1) for k(cat) compared to 7.3 microM and 7.2 min for the wild-type enzyme. By altering these four amino acids, the specificity constants for NADH and NADPH are almost exactly reversed in the mutant relative to the wild type.
Rane
M J
MJ
University of Louisville School of Medicine, Health Sciences Center, Kentucky 40292, USA.
Calvo
K C
KC
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D050540:Ketol-Acid Reductoisomerase; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D017931:DNA Primers; D009249:NADP; D000429:Alcohol Oxidoreductases; D050540:Ketol-Acid Reductoisomerase