Abstract
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 Å. 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.
Highlights
Erally have several [1]
Phylogenetic tree analysis indicates that fungal Pos5 homologs are distinguishable from other NAD kinase (NADK), including Utr1, Yef1, and human NADK, and from NADK3 [6]
On the basis of the tertiary structure of Ppnk complexed with NADϩ (Ppnk-NADϩ), we have demonstrated that Arg-170 of YfjB is one structural determinant that confers a strict specificity for NADϩ alone
Summary
Plasmids—The plasmid pMK2159 carries POS5⌬MTS, encoding a Pos in which the N-terminal 16 residues (residues 2–17) have been replaced by an N-terminal sequence, 29984 JOURNAL OF BIOLOGICAL CHEMISTRY. The plasmid pMK2784 carries cDNA encoding human NADK lacking its N-terminal 87 residues, cloned into the BamHI site of pQE-30 (Qiagen) [12]. Expression and Purification—Pos was expressed in MK2162 cells (E. coli RosettaBlue(DE3) (Novagen) carrying pMK2159) as described previously [6]. Mutated Pos was expressed in E. coli RosettaBlue(DE3) and purified as described above. NADK activity was assayed in a 1.0-ml reaction mixture containing 2.0 mM NADϩ, 10 mM ATP, 10 mM MgCl2, 5.0 mM glucose 6-phosphate, 0.5 unit glucose 6-phosphate dehydrogenase, and 200 mM Tris-HCl, pH 8.0 [6]. NADHK activity was assayed as described [6] in a 1.0-ml reaction mixture containing 2.0 mM NADH, 5.0 mM ATP, 5.0 mM MgCl2, and 100 mM TrisHCl, pH 8.0. Data Collection—Crystals of Pos5-NADH on a nylon loop (Hampton Research) were placed directly in a cold nitrogen-gas
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