Reversal of the nucleotide specificity of ketol acid reductoisomerase by site-directed mutagenesis identifies the NADPH binding site.
Reversal of the nucleotide specificity of ketol acid reductoisomerase by site-directed mutagenesis identifies the NADPH binding site.
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DOI：
10.1006/abbi.1996.9802
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发表时间：
1997-02
影响因子：
3.9
通讯作者：
M. Rane;K. Calvo
中科院分区：
文献类型：
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作者：
M. Rane;K. Calvo
对已发表的七种生物体酮醇酸还原异构酶 (KARI) 的氨基酸序列进行分析，确定了三个具有高度保守序列的区域。这些区域之一预计是 NADPH 结合的二核苷酸折叠。为了确认该区域确实包含NADPH结合位点，我们使用寡核苷酸介导的定点诱变来研究该区域中特定氨基酸与NADPH相互作用的功能。四个带正电荷的氨基酸 R68、K69、K75 和 R76 分别以不同的组合进行单独突变，最后作为四联突变，以评估与 NADPH 的静电相互作用。每个精氨酸单独突变为谷氨酰胺会导致 NADPH 的 k(cat)/K(m) 降低 60 至 100 倍。每个赖氨酸的单独突变不会显着改变与 NADPH 相关的稳态动力学参数。这些突变均不会显着改变酶对 NADH 的亲和力。在研究了这四个氨基酸的双突变后，我们构建了四联体突变体 R68DK69LK75VR76D。该突变体的 NADH 的 K(m) 和 k(cat) 值为 19.3 microM 和 5.3 min(-1)，而野生型酶的 K(m) 和 k(cat) 值为 207 microM 和 0.11 min(-1)。对于四联体突变体，NADPH 的相应值分别为 >200 microM（K(m)）和 2 分钟（-1）（k(cat)），而野生型酶的相应值分别为 7.3 microM 和 7.2 分钟。通过改变这四个氨基酸，相对于野生型，突变体中 NADH 和 NADPH 的特异性常数几乎完全相反。
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.