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

Abstract

The isolation of mutants defective in adenine metabolism in Bacillus subtilis has provided a tool that has made it possible to investigate the role of adenine deaminase in adenine metabolism in growing cells. Adenine deaminase is the only enzyme that can deaminate adenine compounds in B. subtilis, a reaction which is important for adenine utilization as a purine and also as a nitrogen source. The uptake of adenine is strictly coupled to its further metabolism. Salvaging of adenine is inhibited by the stringent response to amino acid starvation, while the deamination of adenine is not. The level of adenine deaminase was reduced when exogenous guanosine served as the purine source and when glutamine served as the nitrogen source. The enzyme level was essentially the same whether ammonia or purines served as the nitrogen source. Reduced levels were seen on poor carbon sources. The ade gene was cloned, and the nucleotide sequence and mRNA analyses revealed a single-gene operon encoding a 65-kDa protein. By transductional crosses, we have located the ade gene to 130؇ on the chromosomal map.When preformed purines are present in the growth medium of Bacillus subtilis, they are readily taken up by specific transport systems and used for nucleotide synthesis. Both adenine, guanine, and hypoxanthine and their nucleoside derivatives serve as sole purine sources, indicating efficient interconversion pathways. Under these conditions, de novo purine synthesis is shut down (37). Adenine is transported by a low-affinity and by a high-affinity transport system (K m ϭ 3 M); the latter system is important when the concentration of adenine is low (4). A key reaction in adenine salvage is the phosphoribosylation of adenine to AMP (see Fig. 1), a reaction that is common to all organisms. However, the conversion of adenine to GMP involves several steps and also different pathways. In Escherichia coli, Salmonella typhimurium (34), and Bacillus cereus (17), adenine is converted to hypoxanthine, with the intermediate formation of adenosine and inosine catalyzed by purine nucleoside phosphorylase and adenosine deaminase. The resulting hypoxanthine then reacts with 5-phosphoribosyl-␣-1-PP i (PRPP) to form IMP, which is converted to GMP via xanthosine monophosphate. 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. This reaction has not yet been demonstrated in bacteria (30, 31). A completely different interconversion pathway includes the first steps of the histidine biosynthetic pathway and the...

The PurR regulon in Lactococcus lactis – transcriptional regulation of the purine nucleotide metabolism and translational machinery

“…We have previously shown that hypoxanthine addition results in an increase in purine nucleotide pools, accompanied by a slight decrease in the pyrimidine pools and a fourfold decrease in the PRPP pool (Jendresen et al, 2011;Martinussen et al, 2003). Purine addition enhances the growth rate by 5-10 % in the exponential growth phase (Kilstrup et al, 2005), as reported for the Gram-positive bacteria B. subtilis and Streptococcus agalactiae (Nygaard et al, 1996;Rajagopal et al, 2005). An identical increase in growth rate and the same fold regulation were observed by supplying individual purine bases or nucleosides as by adding a mixture of adenine, hypoxanthine and guanosine (results not shown).…”

Bacillus subtilis guanine deaminase is encoded by the yknA gene and is induced during growth with purines as the nitrogen source

“…Nucleic acid manipulations and genetic techniques. Isolation and transformation of chromosomal DNA and plasmids were performed as previously described (Nygaard et al, 1996). Treatment of DNA with restriction enzymes, T4 DNA ligase and alkaline phosphatase was performed as recommended by the supplier (Gibco-BRL).…”

“…2 shows the alignment of the metal-binding motif in five identified deaminases. The metal-binding motif of the yknA gene product belongs to a different class from the one found in the B. subtilis adenine deaminase, which contains a motif also found in human GDEase and other amidohydrolases and aminohydrolases (Nygaard et al, 1996 ;Yuan et al, 1999). Interestingly, a sequence of 78 amino acids, encoded by the B. subtilis yaaJ gene encoding a protein of unknown function, showed the highest amino acid identity (44 %) to the yknA gene.…”

“…This sequence also contained the metal-binding motif of the yknA gene. To test if GDEase activity, like adenine deaminase (Nygaard et al, 1996), was stimulated by a divalent cation, cations (1 mM) known to be cofactors in deaminase reactions, were added : Fe# + , Zn# + , Mn# + , Mg# + , Co# + . None of the ions had any effect on GDEase activity, neither had 1 mM EDTA (data not shown).…”

The catalase activity of diiron adenine deaminase

“…However, it is known that in certain bacteria the deamination of adenine can function as a key step in the purine salvage pathway for formation of guanine nucleotides. 20 Therefore, under conditions of oxidative stress inactivation of ADE would block formation of guanine nucleotides via the purine salvage pathway, which in turn might slow cell growth under conditions of oxidative stress. There are known bacterial transcription factors that use metal-catalyzed oxidation of histidine (via H 2 O 2 ) as a redox sensor for controlling oxidative stress levels in cells.…”