Adenine deaminase and adenine utilization in Saccharomyces cerevisiae

Abstract

Compared with other purine salvage and nitrogen catabolism enzymatic activities, adenine deaminase (adenine aminohydrolase [AAHJ; EC 3.5.4.2) activity in Saccharomyces cerevisiae is uniquely regulated. AAH specific activity is not induced by adenine and is reduced sevenfold when cells are cultivated in medium containing proline in place of ammonium as the sole nitrogen source. Exogenous adenine enters metabolic pathways primarily via the function of either AAH or adenine phosphoribosyltransferase (APRT; EC 2.4.2.7). Exogenous adenosine cannot normally be utilized as a purine source. Strains efficiently utilized adenosine or inosine when grown in pH 4.5 medium containing Triton X-100. A recessive mutation permitting utilization of adenosine or inosine in standard media was isolated. In both situations, growth of purine auxotrophs required either AAH or APRT activity. With medium containing either ammonium or proline as a nitrogen source, minimum doubling times of purine auxotrophs deficient in either APRT or AAH were measured. In proline-based medium, AAH and APRT permitted equal utilization of exogenous adenine. In ammonium-based medium, the absence of APRT increased the minimum doubling time by 50%. Similar experiments using sufficient exogenous histidine to feedback inhibit histidine biosynthesis failed to affect the growth rates of adenine auxotrophs blocked in AAH or APRT, indicating that the histidine-biosynthetic pathway does not play a significant role in adenine utilization. The gene that encodes AAH in S. cerevisiae was isolated by complementation using yeast strain XD1-1, which is deficient in AAH, APRT, and purine synthesis. A 1.36-kb EcoRI-SphI fragment was demonstrated to contain the structural gene for AAH by expressing this DNA in Escherichia coli under control of the trp promoter-operator. Northern (RNA) studies using the AAH-, APRT-, and CDC4-coding regions indicated that AAH regulation was not mediated at the level of transcription or mRNA degradation.Purine salvage and interconversion pathways are ubiquitous in cellular metabolism (22,23,39). Genetic deficiency in these pathways results in human diseases, most notably, severe combined immune deficiency, which is the consequence of adenosine deaminase (ADA; EC 3.5.4.4) or purine nucleoside phosphorylase (EC 2.4.2.1) deficiency. These enzymes constitute one of two pathways in humans that mediate the balance of dGTP and dATP in circulating T cells (25).Research aimed at treating these disorders has focused on microorganisms as model systems for understanding the functional importance and regulation of the biochemical pathways. Purine salvage, interconversion, and degradation pathways in Saccharomyces cerevisiae (Fig. 1) have been studied at both the genetic (3, 8, 28, 34-36) and physiological (3, 5, 9, 21, 31, 36, 37) levels. Adenine or hypoxanthine (but not adenosine, inosine, or guanine) (27) can serve as an exogenous purine source and is readily transported into cells by the product of the APP1 gene (26). Adenine enters yeast metabolism (5, ...

Glucose-dependent, cAMP-mediated ATP efflux from Saccharomyces cerevisiae

“…It has been shown, however, that adenine-requiring strains can satisfy the adenine requirement with adenosine if it gains access to the cytoplasm by detergent solubilization of the plasma membrane or by mutation, supporting the view that yeast cells do not normally contain an uptake mechanism for adenine nucleotides (Anderson & Roth, 1976;Deeley, 1992). One exception to this generalization is the finding that sporulating yeast cells apparently utilize extracellular nucleotides and these compounds are required for adenine auxotrophs to undergo sporulation (Jakubowski & Goldman, 1988).…”

“…The increased appearance of extracellular ATP, caused by proton ionophores and diethylstilbestrol, is likely due to a stimulation of efflux rather than to interference with an ATP uptake mechanism because yeast cells are known not to have adenine nucleotide uptake activity (Anderson & Roth, 1976;Deeley, 1992). It has been shown, however, that adenine-requiring strains can satisfy the adenine requirement with adenosine if it gains access to the cytoplasm by detergent solubilization of the plasma membrane or by mutation, supporting the view that yeast cells do not normally contain an uptake mechanism for adenine nucleotides (Anderson & Roth, 1976;Deeley, 1992).…”

“…These results indicate that the release of ATP, and its stimulation by proton ionophores, is a general phenomenon in yeast, and not limited to cells lacking periplasmic acid phosphatases. Furthermore, since extracellular adenosine is not metabolized or taken up by yeast cells (Anderson & Roth, 1976;Deeley, 1992) then play a physiological role in addition to any role played by ATP.…”

The use of amides and other organic nitrogen sources by the phytoplankton Emiliania huxleyi

“…It is not clear if the cascade is at the level of transport or at the level of degradation. Adenine utilization requires an additional enzyme (adenine deaminase) to convert adenine to hypoxanthine (Deeley 1992), which could be induced after enzymes for hypoxanthine utilization; alternatively, adenine transport may be induced after hypoxanthine transport. There is evidence from Chlamydomonas that adenine, guanine, and hypoxanthine are transported by the same porter, but growth on adenine still lags that of guanine at least partly due to a 50% slower maximum transport rate (Lisa et al 1995).…”

Role of adenine deaminase in purine salvage and nitrogen metabolism and characterization of the ade gene in Bacillus subtilis

“…The direct deamination of adenine occurs only in bacteria and lower eukaryotes and has been reported for B. subtilis (15), Azotobacter vinelandii (19), E. coli (24), Candida utilis (19), Schizosaccharomyces pombe (40), Saccharomyces cerevisiae (14, 53), Crithidia fasciculata, and four Leishmania species (23). Adenine deamination is an essential step in the utilization of adenine as the total purine source in S. pombe (40) and S. cerevisiae (14). An alternative and possible route is the direct deamination of AMP catalyzed by AMP deaminase, with ATP as an allosteric activator and GTP as an inhibitor.…”