Identification of ATP‐NADH kinase isozymes and their contribution to supply of NADP(H) in Saccharomyces cerevisiae

Abstract

The genomic DNA sequence of the widely studied yeast Saccharomyces cerevisiae, which is a model organism for eukaryotic cells, contains three NAD kinase homologues, namely, Utr1p, Pos5p and Yel041wp [1][2][3]. NAD kinase (EC 2.7.1.23) catalyses NAD phosphorylation by using phosphoryl donors (ATP or inorganic polyphosphate [poly(P)]), constituting the last step of the NADP biosynthetic pathway [4,5] 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°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 b-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.Abbreviations CgLEU2, LEU2 of yeast Candida glabrata; FOA, 5-fluoroorotic acid; GFP, green fluorescent protein; KNDE, 10 mM potassium phosphate, pH 7.0, containing 0.1 mM NAD, 0.5 mM dithiothreitol and 1.0 mM EDTA; poly(P), inorganic polyphosphate; ScLEU2, LEU2 of yeast Saccharomyces cerevisiae; SD, synthetic dextrose; SG, synthetic glycerol; SD+FOA+Ura, synthetic dextrose ⁄ 5-fluoroorotic acid ⁄ uracil; WT, wild type; YPD, yeast extract ⁄ peptone ⁄ dextrose; YPG, yeast extract ⁄ peptone ⁄ glycerol.

Rewiring of Signaling Networks Modulating Thermotolerance in the Human Pathogen Cryptococcus neoformans

“…In S. cerevisiae, there are three NAD kinases, Utr1, Yef1, and Pos5 (Zhang 2015). Utr1 and Yef1 are cytosolic and Pos5 is a mitochondrial NAD kinase (Kawai et al 2001;Outten and Culotta 2003;Shi et al 2005). UTR1 and POS5 were induced more than twofold during temperature upshift and required for growth at high temperature in C. neoformans (Table 1).…”

Synthetic Lethal and Biochemical Analyses of NAD and NADH Kinases in Saccharomyces cerevisiae Establish Separation of Cellular Functions

“…In yeast, Utr1 was originally reported as a transcript of unknown function (8) and then shown to encode a component of ferrireductase (9) with NAD kinase activity (10). Although the utr1 deletion mutant was recently shown to exhibit poor growth in low iron media (11) consistent with the function of Utr1 within ferrireductase (9), a more general phenotype has not been reported.…”

“…Because two of three NAD/NADH kinase genes had been analyzed with respect to phenotypes and biochemical characteristics, we wished to perform a genetic and biochemical analysis of the remaining NAD/NADH kinase gene in yeast and to analyze phenotypes of double and triple mutants. Recently, Shi et al (11) purified the third yeast NAD/NADH kinase, Yef1, and concluded, as we do, that Yef1 is a cytosolic NAD kinase. However, they claim to produce viable cells deleted for pos5, utr1, and yef1 and suggested the existence of an additional NAD kinase unrelated to the sequence of NAD/NADH kinases (11).…”

“…Recently, Shi et al (11) purified the third yeast NAD/NADH kinase, Yef1, and concluded, as we do, that Yef1 is a cytosolic NAD kinase. However, they claim to produce viable cells deleted for pos5, utr1, and yef1 and suggested the existence of an additional NAD kinase unrelated to the sequence of NAD/NADH kinases (11). In contrast, we report that pos5 utr1 is a synthetic lethal combination, with or without the deletion of yef1.…”

The power to reduce: pyridine nucleotides – small molecules with a multitude of functions

“…The catalytic activity of several NADKs is inhibited by the reduced pyridine nucleotides [99,117,128,129], by high concentrations of NADP + [117,130] or by HgCl 2 [128][129][130]. Interestingly, NADPH, NADH and NADP + inhibit S. cerevisiae NADKs to different extents suggesting a possible regulation by these compounds [129].…”