JOURNAL OF BACTERIOLOGY, Apr. 1983, p. 192-199 Vol. 154, No. 1 0021-9193/83/040192-08$02.00/0 Copyright C 1983, American Society for Microbiology Purine and Glycine Metabolism by Purinolytic Clostridia PETER DURREt AND JAN R. ANDREESEN* Institut fur Mikrobiologie, Universitat Gotingen, D-3400 Gottingen, Federal Republic of Germany Received 30 August 1982/Accepted 12 January 1983 Cell extracts of Clostridium acidiurici, C. cylindrosporum, and C. purinolyti- cum converted purine, hypoxanthine, 2-hydroxypurine, 6,8-dihydroxypurine, and uric acid into xanthine by the shortest possible route. Adenine was transformed to xanthine only by C. purinolyticum, whereas the other two species formed 6- amino-8-hydroxypurine, which was neither deaminated nor hydroxylated further. 8-Hydroxypurine was formed from purine by all three species. Xanthine dehy- drogenase activity was constitutively expressed by C. purinolyticum. Due to the lability of the enzyme activity, comparative studies could not be done with a purified preparation. All enzymes reported to be involved in formiminoglycine metabolism of C. acidiurici and C. cylindrosporum were present in C. purinolyti- cum. However, glycine was reduced directly to acetate in all three species, as indicated by radiochemical data and by the detection of glycine reductase in cell extracts of C. cylindrosporum and C. purinolyticum. The expression of glycine reductase and the high ratio of glycine fermented to uric acid present points to an energetic advantage for the glycine reductase system, which is expressed when selenium compounds are added to the growth media. Purines are important compqrnents of nucleic central position in purine degradation (14). In acids and nucleotides. However, free purine contrast, selenium-supplemented cells oxidize bases can be quite toxic for bacteria and eucary- hypoxanthine directly to xanthine (15), and not otes (19). A variety of bacteria transform or via 6,8-dihydroxypurine and uric acid as report- degrade purines (43). The enzyme xanthine de- ed for C. cylindrosporum (6). The hydrolysis of hydrogenase, a molybdo-iron-sulfur flavopro- xanthine to formiminoglycine occurs in C. puri- tein, is generally responsible for the transforma- nolyticum via the same intermediates as those tion of the purine ring system. Its substrate reported for C. cylindrosporum (15, 24). How- specificity and pattern of hydroxylation depend ever, the metabolism of glycine seems to be on the organism involved (11). Recently, the different: C. acidiurici and C. cylindrosporum trace element selenium was found to be impor- convert glycine via serine and pyruvate to ace- tant for the expression of an active xanthine tate (9, 28, 31, 44), whereas C. purinolyticum dehydrogenase in bacteria specialized in anaero- reduces glycine directly to acetate by the action bic purine degradation (13, 46). The three known of one enzyme, glycine reductase, which re- species, Clostridium acidiurici, C. cylindro- quires selenium compounds (16, 36). Therefore, sporum, and C. purinolyticum, are phenotypi- purine and glycine metabolism by C. acidiurici cally very similar, but genotypically quite dis- and C. cylindrosporum were reexamined. The tinct. The spectrum of purines utilized by each results obtained by using selenium-supplement- of these bacteria for growth differs slightly (13). ed cells revise the hydroxylation pattern of In a study on xanthine dehydrogenase of C. purine compounds and of glycine metabolism cylindrosporum (6), a preference of the enzyme compared with previous data. Thus, the avail- for attacking position 8 of the purine ring system ability of selenium influences the substrate was found, and uric acid was concluded to be a specificity and the flow of purine carbon in these central intermediate in purine transformation of organisms by the formation of more active and this organism. The purified xanthine dehydroge- favorable enzymes. nase used was, however, much less active than crude extracts prepared from selenium-supple- MATERIALS AND METHODS mented cells (46). Organisms and growth media. C. purinolyticum Recent studies with C. purinolyticum showed WA-1 (DSM 1384) was grown on adenine as described that in selenium-deprived cells uric acid has a previously (13). C. acidiurici 9a (ATCC 7906, DSM 604) and C. cylindrosporum HC-1 (ATCC 7905, DSM t Present address: Department of Biochemistry, University 605) were obtained from the Deutsche Sammlung von of California, Berkeley, CA 94720. Mikroorganismen, Gottingen, Federal Republic of 192 VOL. 154, 1983 CLOSTRIDIAL PURINE AND GLYCINE DEGRADATION 193 Germany, and were cultured in a similar medium with droxypurine was additionally established by cochro- adenine replaced by uric acid (10 mM). In some matography with the actual substance, kindly donated experiments, glycine (100 mM) was added. C. sticklan- by H. A. Barker, University of California, Berkeley. dii (ATCC 12662, DSM 519) was kindly provided by Attempts to prepare 3-(lH-pyrazolo[3,4-dlpyrimidine- A. C. Schwartz, Institute of Botany, University of 4-ylamino)-1-propyl-6-aminohexanoate (=compound Bonn, Federal Republic ofGermany, and was cultivat- III) for affinity chromatography of xanthine dehydro- ed according to Stadtman (36). All media were pre- genase as described previously (17) proved to be pared under strictly anaerobic conditions (13). unsuccessful. However, the substance could be pre- Analytcl methods. Acetate was determined by an pared by changing the sequence ofadditions during the enzymatic procedure (12), formate and glycine by synthesis. It was absolutely necessary to add N,N'- colorimetric methods (22, 33). Identification and quan- carbonyldiimidazole in advance of compound I. Com- tification of purines were performed by means of high- pound III synthesized in this way proved to be identi- pressure liquid chromatography on a reversed-phase cal with material kindly provided by G. Blankenhorn, (C18) column, using potassium phosphate buffer (100 University of Konstanz, Federal Republic of Germa- mM, pH 2.7 to 3.7) as eluent (15). 8-Hydroxypurine ny. Tetrahydrofolic acid was kindly provided by eluted in advance of the unsubstituted purine. Lynne Quandt and M. Braun, University ofGottingen, Tracer experiments. Fermentation of radioactive Federal Republic of Germany. Formiminoglycine-p- glycine in the presence of uric acid by resting cells of phenylethylester hydrochloride was synthesized ac- C. acidiurici and C. cylindrosporum was performed in cording to Freter et al. (18). Polymin P used for principle as already described (16). In this case, the purification of ferredoxin (35) was donated by BASF, reaction buffer had the same composition as the Ludwigshafen, tederal Republic of Germany. All oth- growth medium except that glycine, potassium bicar- er chemicals used were of the highest purity commer- bonate, and yeast extract were omitted and 0.15 ILM cially available. thiamine was added. Enzyme assays. Cell extracts were prepared by sus- RESULTS pending 1 g of cells (wet weight, harvested at the end of the exponential-growth phase) in 2 ml of potassium Purine interconversion. Cell extracts of C. phosphate buffer (10 mM, pH 7.1); adding 1 mg of purinolyticum, C. acidiurici, and C. cylindro- lysozyme, 0.1 mg of DNase, and 1 mM dithioerythri- sporum were allowed to act on buffered purine tol; and incubating anaerobically for 1 h at 37°C. solutions (4 mM potassium phosphate buffer, pH Subsequently, the suspension was passed once through a chilled French pressure cell at 1,050 kPa/ 7.5) in the presence of 6 mM EDTA. This agent cm2. After centrifugation at 40,000 x g (30 min) and does not inhibit purine interconversion but pre- 4°C, the supernatant was kept at 0°C under an atmo- vents purine breakdown (15), which generally sphere of nitrogen in the dark and immediately used starts from xanthine (24). Xanthine was found to for enzymatic assays. Cells for the glycine reductase be a transformation product of all purine com- assay were stored according to Barnard and Akhtar pounds tested except for adenine in the case of (4). The protein content of the extracts was deter- C. acidiurici and C. cylindrosporum (Table 1). mined by the biuret method (5). All enzymatic tests Unlike C. purinolyticum, these organisms were performed under strictly anaerobic conditions seemed to be unable to deaminate adenine to at 37°C. The following assays were used: xanthine de- hydrogenase (13), carbamate kinase (34), glycine for- hypoxanthine; instead, adenine was hydroxylat- miminotransferase (40), methenyltetrahydrofolate cy- ed to yield 6-amino-8-hydroxypurine. 6-Amino- clohydrolase (23, 41), methylenetetrahydrofolate 2-hydroxypurine (isoguanine) and 6-amino-2,8- dehydrogenase (23, 42), formyltetrahydrofolate syn- dihydroxypurine were not detected as products. thetase (23, 27), L-serine dehydratase (16) (activation The unsubstituted purine ring was hydroxylated of extract according to Carter and Sagers [8]), serine in positions 6 and 8. However, 8-hydroxypurine hydroxymethyltransferase (4), glycine decarboxylase was only transiently formed within the first 2 h (32), and glycine reductase (36). One unit of enzyme by C. purinolyticum, whereas its concentration activity was defined as 1 M.mol of substrate trans- did not decrease with time in the extracts of the formed or product formed per min at 37C. About 80%o of the xanthine dehydrogenase activity was lost in cell other two organisms. Xanthine was the only extracts within 3 days at 4°C when 50 mM potassium product detected after incubation of the extracts