Molecular conversion of NAD kinase to NADH kinase through single amino acid residue substitution
Molecular conversion of NAD kinase to NADH kinase through single amino acid residue substitution
复制标题
DOI：
10.1074/jbc.m502518200
复制
发表时间：
2005-06-24
影响因子：
4.8
通讯作者：
Murata, K
中科院分区：
文献类型：
--
作者：
Mori, S;Kawai, S;Murata, K
NAD激酶磷酸化NAD（+）以形成NADP（+），并且对NAD（+）具有严格特异性，而NADH激酶磷酸化NAD（+）和NADH两者，从而显示宽松的底物特异性。基于它们的一级和三级结构，NAD和NADH激酶之间底物特异性的差异被认为是由一个对齐的残基引起的：在5种NADH激酶中的Gly或极性氨基酸（Gln或Thr）和在2种NAD激酶中的带电氨基酸（Arg）。在两种NAD激酶中用Gly取代Arg放松了底物特异性（即将NAD激酶转化为NADH激酶）。在一个NAD激酶中用极性氨基酸取代Arg也放松了底物特异性，而用带电和疏水性氨基酸取代则没有显示出类似的结果。相比之下，在一个NADH激酶中用Arg取代Gly不能将其转化为NAD激酶。这些结果表明，在感兴趣的位置上的带电或疏水性氨基酸残基对于NAD激酶对NAD（+）的严格特异性是至关重要的，而Gly或极性氨基酸残基不是NADH激酶的松弛底物特异性的唯一决定因素。本文还讨论了207个NAD激酶同源物中该位置残基保守性的意义。
NAD kinase phosphorylates NAD(+) to form NADP(+) and is strictly specific to NAD(+), whereas NADH kinase phosphorylates both NAD(+) and NADH, thereby showing relaxed substrate specificity. Based on their primary and tertiary structures, the difference in the substrate specificities between NAD and NADH kinases was proposed to be caused by one aligned residue: Gly or polar amino acid (Gln or Thr) in five NADH kinases and a charged amino acid (Arg) in two NAD kinases. The substitution of Arg with Gly in the two NAD kinases relaxed the substrate specificity (i.e. converted the NAD kinases to NADH kinases). The substitution of Arg in one NAD kinase with polar amino acids also relaxed the substrate specificity, whereas substitution with charged and hydrophobic amino acids did not show a similar result. In contrast, the substitution of Gly with Arg in one NADH kinase failed to convert it to NAD kinase. These results suggest that a charged or hydrophobic amino acid residue in the position of interest is crucial for strict specificity of NAD kinases to NAD(+), whereas Gly or polar amino acid residue is not the sole determinant for the relaxed substrate specificity of NADH kinases. The significance of the conservation of the residue at the position in 207 NAD kinase homologues is also discussed.