Identification of ATP-NADH kinase isozymes and their contribution to supply of NADP(H) in Saccharomyces cerevisiae.
ATP-NAD kinase phosphorylates NAD to produce NADP by using ATP, whereas ATP-NADH kinase phosphorylates both NAD and NADH. Three NAD kinase homologues, namely, ATP-NAD kinase (Utr1p), ATP-NADH kinase (Pos5p) and function-unknown Yel041wp (Yef1p), are found in the yeast Saccharomyces cerevisiae. In this study, Yef1p was identified as an ATP-NADH kinase. The ATP-NADH kinase activity of Utr1p was also confirmed. Thus, the three NAD kinase homologues were biochemically identified as ATP-NADH kinases. The phenotypic analysis of the single, double and triple mutants, which was unexpectedly found to be viable, for UTR1, YEF1 and POS5 demonstrated the critical contribution of Pos5p to mitochondrial function and survival at 37 degrees C and the critical contribution of Utr1p to growth in low iron medium. The contributions of the other two enzymes were also demonstrated; however, these were observed only in the absence of the critical contributor, which was supported by complementation for some pos5 phenotypes by the overexpression of UTR1 and YEF1. The viability of the triple mutant suggested that a 'novel' enzyme, whose primary structure is different from those of all known NAD and NADH kinases, probably catalyses the formation of cytosolic NADP in S. cerevisiae. Finally, we found that LEU2 of Candida glabrata, encoding beta-isopropylmalate dehydrogenase and being used to construct the triple mutant, complemented some pos5 phenotypes; however, overexpression of LEU2 of S. cerevisiae did not. The complementation was putatively attributed to an ability of Leu2p of C. glabrata to use NADP as a coenzyme and to supply NADPH.
Department of Basic and Applied Molecular Biotechnology, Division of Food and Biological Science, Graduate School of Agriculture, Kyoto University, Uji, Kyoto, Japan.
Feng
F
Shi
Shigeyuki
S
Kawai
Shigetarou
S
Mori
Emi
E
Kono
Kousaku
K
Murata
D050539:3-Isopropylmalate Dehydrogenase; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism; D041221:Candida glabrata / Q000201:enzymology; D002470:Cell Survival; D003600:Cytosol / Q000201:enzymology; D005816:Genetic Complementation Test; D007501:Iron / Q000378:metabolism; D007527:Isoenzymes; D008928:Mitochondria / Q000201:enzymology / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism*; D010641:Phenotype; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013379:Substrate Specificity
D007527:Isoenzymes; D029701:Saccharomyces cerevisiae Proteins; D009243:NAD; D009249:NADP; D000255:Adenosine Triphosphate; D007501:Iron; D000429:Alcohol Oxidoreductases; D050539:3-Isopropylmalate Dehydrogenase; D017853:Phosphotransferases (Alcohol Group Acceptor); C021844:NAD kinase