Adenine aminohydrolase: occurrence and possible significance in trypanosomid flagellates.

Abstract

Adenine aminohydrolase (EC 3.5.4.2) from four species of Leishmania and from Crithidia fasciculata was examined for specific activities, affinity for substrate (adenine), and stability to heat. All were found to be strongly and noncompetitively inhibited by both coformycin and deoxycoformycin, two tight-binding inhibitors-of adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4). Deoxycoformycin is the more potent inhibitor of the two. Neither inhibitor was active against the purine phosphoribosyltransferases. When deoxycoformycin was added to the defined growth medium containing hypoxanthine as the purine source, the. growth of C. fasciculata was unaffected, but when adenine was the purine source for the organism, severe inhibition resulted. This implies that hypoxanthine is the obligatory base for nucleotide synthesis and that the adenine phosphoribosyltransferase (AMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.7) is, in some manner, denied access to exogenous substrate. Since our earlier report (1) on the occurrence and properties of adenine aminohydrolase (EC 3.5.4.2) (adenine deaminase) in the trypanosomid flagellate Crithidia fasciculata, the enzyme has been reported from Leishmania tropica (2), L. donovani, and L. braziliensis (3). We have extended our investigations of this somewhat unusual enzyme and have found it to be present also in L. tarentolae and L. mexicana, and have shown that it is subject to drastic inhibition by two tight-binding adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) inhibitors, coformycin and deoxycoformycin. By the use of these potent inhibitors, it has been possible to assess to some degree the role of the enzyme as a control mechanism in these purine-requiring organisms. MATERIALS AND METHODS[8-14C]Adenine and [8-14C]hypoxanthine (both at 50 Ci/mmol, 1 Ci = 3.70 X 1010 becquerels) were obtained from Amersham/Searle. All chemicals of the defined medium used for C. fasciculata were obtained from Sigma; the ingredients of the semi-defined medium of Berens et al. (4) C. fasciculata, a mosquito parasite, is the strain that has been used in this laboratory for the past 20 years (5-6). L. tarentolae, a lizard parasite, was obtained through the courtesy of Larry Simpson (University of California, Los Angeles). This is the strain originally used by Trager (7). L. donovani, L. brazilhensis, and L. mexicana, all human pathogens, were kindly supplied by J. J. Marr (St. Louis University). These strains have been used by Marr and his associates for the past several years (3).-C. fasciculata was grown in the defined medium of Kidder and Dutta (5), in low profile flasks when large amounts of tissue were needed or in side-arm Nephelo flasks (Bellco, Vineland, NJ) or 25 X 125 mm optically clear tubes for the growth studies.Growth was followed by reading optical densities at 650 nm, with uninoculated medium as the standard. In all cases hemin, glucose, and inhibitors (if any) were filter sterilized and added to the bulk of the heat-sterilized medium.L. tarentolae w...

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

“…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).…”

Inhibition of growth and purine-metabolizing enzymes of trypanosomid flagellates by N6-methyladenine

“…Our earlier report (18) has shown this to be an important enzyme in the purine pathway of C. fasciculata and leishmaniae (Fig. 3).…”

“…The pathways of purine nucleotide interconversions in these parasitic organisms resemble those found in mam n cells (see Fig. 3) except for the absence of de novo purine base biosynthesis (7,21) and, for Crithidia fasciculata and the leishmaniae, the presence of adenine-deaminating activity (16,18,21). For these reasons, the purine salvage pathway has become popular as a target for chemotherapeutic investigations in these organisms.…”

Amplification of Adenine Phosphoribosyltransferase Suppresses the Conditionally Lethal Growth and Virulence Phenotype of Leishmania donovani Mutants Lacking Both Hypoxanthine-guanine and Xanthine Phosphoribosyltransferases

“…The construction and characterization of a conditionally lethal ⌬hgprt/⌬xprt null mutant using targeted gene replacement approaches that exhibit patently atypical growth requirements provided powerful genetic evidence for the hypothesis that all salvage of purine nucleobases and nucleosides by L. donovani ultimately occurs through HGPRT or XPRT and that APRT and adenosine kinase are functionally redundant (10). Whereas wild type L. donovani can proliferate in virtually any purine nucleobase or nucleoside (2,3,11), the ⌬hgprt/⌬xprt mutant exhibits an absolute requirement for adenine or adenosine as a purine source and 2Ј-deoxycoformycin (dCF), an inhibitor of the leishmanial adenine aminohydrolase enzyme (10,12). Unlike wild type L. donovani, the ⌬hgprt/⌬xprt parasites cannot grow without 2Ј-deoxycoformycin or with hypoxanthine, guanine, xanthine, guanosine, inosine, or xanthosine as the sole purine nutrient (10).…”