Structural Determinants of Discrimination of NAD+ from NADH in Yeast Mitochondrial NADH Kinase Pos5
Structural Determinants of Discrimination of NAD+ from NADH in Yeast Mitochondrial NADH Kinase Pos5
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DOI：
10.1074/jbc.m111.249011
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发表时间：
2011-08-26
影响因子：
4.8
通讯作者：
Murata, Kousaku
中科院分区：
文献类型：
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作者：
Ando, Takuya;Ohashi, Kazuto;Murata, Kousaku
NAD激酶催化NAD（+）磷酸化合成NADP(+)，而NADH激酶催化NADH转化为NADPH。酿酒酵母线粒体蛋白Pos5显示出比NAD激酶更高的NADH激酶活性，因此被称为NADH激酶。为了阐明Pos5高NADH激酶活性的结构决定因素及其对NADH的选择性，我们在2.0埃的分辨率下测定了Pos5与NADH络合的三级结构。将Pos5的三级结构与人类和细菌NAD激酶的结构进行比较，发现Pos5的Arg-293与人类NAD激酶的His-351相对应，在nadh结合位点表面带正电荷，而相应的His残基则不带正电荷。因此，Arg-293转化为His残基将NADH激酶活性与NAD激酶活性之比从8.6降至2.1。相反，人NAD激酶的Ala-330和His-351同时转变为Ser和Arg残基，使NADH激酶活性与NAD激酶活性之比从0.043提高到1.39；人Ala-330对应于与Arg-293侧链相互作用的Pos5 Ser-272。Arg-293和Ser-272在Pos5同源物（推定的NADH激酶）中高度保守，但在推定的NAD激酶中不保守。因此，Pos5的Arg-293是NADH选择性的主要决定因素。此外，Ser-272似乎有助于Arg-293获得适当的构象。
NAD kinase catalyzes the phosphorylation of NAD(+) to synthesize NADP(+), whereas NADH kinase catalyzes conversion of NADH to NADPH. The mitochondrial protein Pos5 of Saccharomyces cerevisiae shows much higher NADH kinase than NAD kinase activity and is therefore referred to as NADH kinase. To clarify the structural determinant underlying the high NADH kinase activity of Pos5 and its selectivity for NADH over NAD(+), we determined the tertiary structure of Pos5 complexed with NADH at a resolution of 2.0 angstrom. Detailed analysis, including a comparison of the tertiary structure of Pos5 with the structures of human and bacterial NAD kinases, revealed that Arg-293 of Pos5, corresponding to His-351 of human NAD kinase, confers a positive charge on the surface of NADH-binding site, whereas the corresponding His residue does not. Accordingly, conversion of the Arg-293 into a His residue reduced the ratio of NADH kinase activity to NAD kinase activity from 8.6 to 2.1. Conversely, simultaneous changes of Ala-330 and His-351 of human NAD kinase into Ser and Arg residues significantly increased the ratio of NADH kinase activity to NAD kinase activity from 0.043 to 1.39; human Ala-330 corresponds to Pos5 Ser-272, which interacts with the side chain of Arg-293. Arg-293 and Ser-272 were highly conserved in Pos5 homologs (putative NADH kinases), but not in putative NAD kinases. Thus, Arg-293 of Pos5 is a major determinant of NADH selectivity. Moreover, Ser-272 appears to assist Arg-293 in achieving the appropriate conformation.