Vol. 131, No. 3 Printed in U.S.A.
JouRNAL oF BACTRIOLOGY, Sept. 1977, p. 789-794 Copyright 0 1977 American Society for Microbiology
Metabolism of 6-Aminonicotinic Acid in Escherichia colil JOHN R. COBB, SUSAN C. PEARCY, AND R. K. GHOLSON* Department ofBiochemistry, Oklahoma State University, Stillwater, Oklahoma 74074 Received for publication 4 March 1977
A late-log-phase culture of an Escherichia coli nadB pncA double mutant took up 6-[7-"4C]aminonicotinic acid and excreted 6-[14C]aminonicotinamide. This mutant also accumulated intracellularly several radioactive compounds which have been tentatively identified as 6-amino analogs of compounds in the pyridine nucleotide cycle. It is concluded that 6-aminonicotinamide and 6aminonicotinic acid probably exert at least a portion of their bacteriostatic effects by being metabolized, by the enzymes of the pyridine nucleotide cycle, to 6-aminonicotinamide adenine dinucleotide and 6-aminonicotinamide adenine dinucleotide phosphate. These compounds are not electron acceptors and are known inhibitors of some pyridine nucleotide-linked dehydrogenases.
6-Aminonicotinamide (6-ANAm) and 6-aminonicotinic acid (6-ANA) are analogs of nicotinamide and nicotinic acid, respectively. 6-ANA is also a structural analog and antimetabolite ofp-aminobenzoic acid (PABA) (9). The bacteriostasis produced by 6-ANA in Escherichia coli and Streptococcus haemolyticus was reversed by PABA (9). 6-ANA also inhibited multiplication of T2 bacteriophage, with the inhibition being reversed by nicotinamide, PABA, or acetate (23). 6-ANAm was also found to be toxic to mammals, with low doses causing blindness and paralysis in the extremities of rabbits and rats (10). The toxicity of 6-ANAm was reversed by nicotinamide, and it was concluded that 6ANAm is a potent antimetabolite of nicotinamide (11). In rats, 6-ANAm also has a teratogenetic effect, which is prevented by nicotinamide (3). In addition, 6-ANAm has been shown to have strong carcinostatic activity, which is reversed by nicotinamide (18). 6-ANAm was postulated to produce these effects in mammalian systems by conversion to an enzymatically inactive nicotinamide adenine dinucleotide (NAD) analog (11). Dietrich et al. (5) demonstrated the formation of 6-aminonicotinamide adenine dinucleotide (6-ANAD) and 6-aminonicotinamide adenine dinucleotide phosphate (6ANADP) from 6-ANAm in vivo by mouse tissues and in vitro by the exchange reaction catalyzed by pig brain NAD glycohydrolase. 6ANAD was found to inhibit several NADlinked dehydrogenases (5), and 6-ANADP was shown to be a potent inhibitor of 6-phosphogluconate dehydrogenase (13). Since 6-ANAm is 15 times more toxic than 6-ANA in mammals (W. I Journal article J-3291 of the Oklahoma State Agricultural Experiment Station.
789
J. Johnson and J. D. McCall, Fed. Proc., p. 284, 1956), the major pathway for the fonnation of the NAD and NAD phosphate (NADP) analogs in vivo is probably by a direct exchange of 6ANAm into NAD and NADP catalyzed by NAD glycohydrolase. Human and chicken erythrocytes form small quantities of 6-ANAm mononucleotide (6-ANMN) and 6-ANAD from 6ANAm, whereas 6-ANA mononucleotide (6ANaMN), but not 6-ANA adenine dinucleotide (6-ANaAD) or 6-ANAD, is formed from 6-ANA
(4).
The metabolic fate of 6-ANA in bacteria is less well documented. Wacker et al. (22) reported that 6-44C]ANA is incorporated into two radioactive compounds by Enterococcus stei. One was presumed to be a folic acid analog, since its formation was suppressed by addition of PABA to the culture medium, and the other was presumed to be an NAD analog because its formation was suppressed by nicotinic acid, it had the same Rf as NAD in the paper chromatography system used, and it was cleaved to 6ANA by perchloric acid treatment (22). This paper concerns the metabolic fate ofbacteriostatic concentrations of 6-ANA in E. coli. Since a thorough study has failed to show any NAD glycohydrolase activity in E. coli (2), our working hypothesis was that 6-ANAm and 6ANA are metabolized to NAD and NADP analogs by the enzymes of the pyridine nucleotide cycle (6, 14; see Fig. 1). The evidence obtained supports this hypothesis. MATERIALS AND METHODS Bacterial strains. The strains used in this study are niacin-requiring mutants of E. coli K-12 originally obtained from T. K. Sundaram (19) and designated by him W3899 and W3899 NAm 11. W3899 is
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blocked in the de novo pathway of NAD biosynthesis (nadB) (21), and W3899 NAm 11 contains an additional block in the conversion of nicotinamide to nicotinic acid (nadB pncA). Media and growth conditions. Cultures were grown with vigorous shaking or aeration at 37°C in the minimal medium of Yates and Pardee (24) with the additions noted in the tables and figure legends. For labeling experiments bacteria were grown in volumes of 5, 25, or 100 ml. Each culture was inoculated with a log-phase starter culture of 5% of its volume. The bacteria were harvested by centrifugation in a Sorvall RC2-B at 15,000 rpm for 15 min at 4°C in an 58-34 rotor. Chemicals. 6-ANAm was purchased from Aldrich Chemical Co. 6-ANA was prepared by refluxing 6ANAm in 50 weight volumes of 1.0 N NaOH for 1 h. The cooled solution was adjusted to pH 4 with glacial acetic acid, and the white precipitate of 6-ANA was collected and recrystallized from 40% acetic acid. The melting point was 297°C, in agreement with literature values. The product appeared homogeneous by thin-layer chromatography. Potassium [14C]cyanide, Hu2PO4, and [7-14C]nicotinic acid were purchased from New England Nuclear. [83H]adenine was obtained from Amersham/Searle. Nicotinic acid, nicotinamide, and alcohol dehydrogenase were purchased from the Sigma Chemical Co. PABA was purchased from Eastman Organic Chemicals; 2-amino-5-nitro-pyridine was from Aldrich Chemical Co., and 2,5-diphenyloxazole and 1,4bis-(6-phenyloxazolyl)-benzene were from the Packard Instrument Co. All other chemicals were obtained from local supply houses. The synthesis of 6-[7-'4C]ANA was based on a method described by Rath and Prange (17). This synthesis is similar to that described by Wacker et al. (22). 2,5-Diaminopyridine was synthesized from 2-amino-5-nitropyridine according to Rath and Prange (17), and 150 mg was dissolved in 1 ml of 0.8 N HCI in a 10-ml beaker. This solution was diazotized by the addition of 60 mg of NaNO2 dissolved in 0.5 ml of water. The solution was neutralized with solid NaCO, until CO2 evolution stopped and added to a solution of 19 mg of CuCN and 39 mg ofK14CN (1 mCi) in 2 ml of water in a 20-ml beaker. The mixture was heated for 1 h at 40°C. The reaction mixture was then evaporated carefully to dryness on a hot plate, and the solid was transferred to a Soxhlet cup and extracted with ether in a Soxhlet apparatus until no more yellow color was extracted. The ether was evaporated and the yellow solid 2-amino-5-[14Ccyanopyridine was hydrolyzed with 2 ml of 2 N NaOH at 100°C for 1 h. The solution was neutralized with HCI and applied to a Dowex-1 (formate) column (1 by 10 cm), which was washed first with 60 ml of water, which removed a radioactive peak (presumably nitrile), then with 60 ml of 10-4 M formic acid, which eluted a yellow compound, and then with 30 ml of 5 x 10-4 M formic acid. The 6-[7-14C]ANA was then eluted with 2 x 10-3 M formic acid. The fractions containing 6-[4C]ANA were pooled and lyophilized. The yield was 9.4 mg, and the specific activity was 0.96 mCi/mmol. 6-[7-14C]ANAm was prepared by heating the 2-amino-5-[14C]cyanopyridine in 2 ml
of water with Dowex-50 H+ at 100°C for 1 h. 6[U4C]ANAm was removed from the resin with water and purified on a Dowex-1 (formate) column. The radiochemical purity of the 6-[14C]ANA and 6['4C]ANAm was checked by radioautography after thin-layer chromatography in solvent systems I and II (see below). Several experiments to be described were performed with another preparation of 6-[714ClANA of slightly higher specific radioactivity. Thin-layer chromatography. Thin-layer chromatography was carried out with Analtech glass plates coated with cellulose 250 ,um thick containing fluorescent indicator. Solvent system I was n-butanol saturated with water-NH4OH, 66:6. Solvent system II was n-butanol saturated with water-glacial acetic acid, 66:3. Liquid scintiUation counting. Radioactivity was determined with a Packard Tri-Carb liquid scintillation spectrometer. The scintillation liquid was composed of 4 g of 2,5-diphenyloxazole, 0.2 g of 1,4bis-(5-phenyloxazolyl)-benzene, 400 ml of ethanol, and 600 ml of toluene per liter. The disintegrations per minute of each sample was calculated by the channels ratio method for quench correction and counting efficiency.
RESULTS A preliminary experiment was carried out to determine the gross metabolic fate of 6[14C]ANA in E. coli. Since NAD synthesis in E. coli can occur either by a de novo pathway from aspartate (20) or by utilization of the exogenously supplied base, an nadB mutant, which lacks the former pathway, was used to increase the utilization of the exogenously supplied analog. This strain (W3899 NAm 11) also carries a mutation at the pncA locus, which is the structural gene for nicotinamide deamidase (19). This block in the pyridine nucleotide cycle prevents utilization of nicotinamide; hence, any nicotinamide generated during normal operation of the pyridine nucleotide cycle will not be further metabolized and will accumulate in the medium (Fig. 1). It has been previously shown that this double mutant excretes [14C]nicotinamide into the medium when incubated in the presence of [14C]nicotinic acid (1). Since the only known reaction sequence for this conversion is the amidation of nicotinic acid adenine dinucleotide to NAD and the subsequent degradation of NAD to nicotinamide, the excretion of nicotinamide was considered strong evidence for the presence of a functioning pyridine nucleotide cycle in E. coli (1, 14). When washed cells of W3899 NAm 11 were incubated with 6-[7-14C]ANA for 6 h, two major compounds were recovered from the incubation medium (Fig. 2). The compound eluted from Dowex-1 (formate) with 1 N formic acid (peak II) was identified as unchanged 6-[14C]ANA by thin-layer chromatography in solvent systems I
METABOLISM OF 6-AMINONICOTINIC ACID IN E. COLI
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starting material was found in the medium. When W3899 (nadB) was incubated with these "C-labeled amides, the corresponding acids NA 4-- NAm-NMN were excreted into the medium. These results ACID pnc A establish that 6-ANAm is a substrate for nicoFIG. 1. Pyridine nucleotide cycle in E. coli. nadA, tinamide deamidase. nadB, nadC, and pncA show the metabolic location The deamidation of these amides to the correof known genetic lesions. Abbreviations: DHAP, di- sponding acids, which accumulate in the mehydroxyacetone phosphate; NaMN, nicotinic acid dium of E. coli W3899 (which has an intact mononucleotide; NaAD, nicotinic acid adenine dinunucleotide cycle), suggests that nicocleotide; NMN, nicotinamide mononucleotide; NAm, pyridine tinamide deamidase is unregulated and supnicotinamide; NA, nicotinic acid. ports the conclusion of Imsande and Pardee (8) that the formation of nicotinic acid mononucleotide from nicotinic acid is a rate-limiting 1.0 step in NAD biosynthesis in E. coli. H20 I N Formate , Time course of 6-414CIANA uptake and 6o.s [14C]ANAm excretion' When a culture of E. 0.8 II coli W3899 NAm 11 containing approximately 2 0.7 x 10'1 cells/ml was incubated in minimal me1I 0.6 dium with 2 x 1O-5 M 6-[7-14C]ANA, this com, 1 0.5 pound was taken up continuously by the bacte~~~~~~~~~~~~~~~~~~~~I 0.4 ria for the 6-h duration of the experiment (Fig. A------tI 3). At 90 min after the beginning of incubation 0.3 6-44C]ANAm was detectable in the medium, 0.2 I~~~~~~~ and the amount of this compound present con0.1 tinued to increase linearly for the rest ofthe 6-h experiment. After an initial period the amount 5 I5 25 35 45 55 of total extracellular radioactivity decreased Fraction No. FIG. 2. Dowex-1 (formate) chromatography ofme- continuously over the course ofthe experiment. dium from an E. coli nadB pncA double mutant After 6 h approximately 45% of the radioactivincubated with 6-['4C]ANA. A late-log-phase culture CO
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ofE. coli W3899 NAm 11 was washed in 0.9% saline and suspended at a concentration of approximately 3 x 109 cellslml in 10 ml of minimal medium containing 2 x 10-5 M 6-[14C]ANA (3.2 mCi/mmol) and shaken at 37°C for 6 h. The cells were removed by centrifugation, 1 p,mol each of 6-ANA and 6-ANAm were added, and the medium was placed on a 1- by 35-cm column of Dowex 1-x8 formate, which was eluted with water and 1 N formic acid. Fractions of 4 ml were collected, and absorbance at 260 nm (A2nd) and radioactivity were determined.
15 13 11I
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9
7 7
5 3
and II. The compound eluted with water (peak I) was identified as 6-['4C]ANAm by chromatography in these solvent systems and by hydrolysis to 6-14C]ANA in 1 N NaOH. This conversion of 6-ANA to 6-ANAm by W3899 NAm 11 provides strong presumptive evidence that the 6-ANA is converted to pyridine nucleotide analogs and then to 6-ANAm by the reactions of the pyridine nucleotide cycle. Control experiments were carried out to show that the pncA mutant did not excrete "4C-labeled compounds upon incubation with 6-[74CIANAm or [7-14C]nicotinamide. When W3899 NAm 11 cells (nadB pncA) were incubated with 6-[14C]ANAm or [14C]nicotinamide under the conditions described above, only the
30
90 120
180 240 300 360 Time (min)
FIG. 3. Uptake of 6-[14C]ANA and excretion of 6['4C]ANAm by an nadB pncA mutant as a function of time. A 100-ml amount of medium containing 2 x 10-5 M 6-[7-14CIANA (32 mCilmmol) was inoculated with approximately 2 x 1011 cells harvested
from a late-log-phase culture of E. coli W3899 NAm 11. One-milliliter samples were removed at the times indicated, and the bacteria were separated by centrifugation. The supernatant solutions were analyzed for 6-P14CJANA and 6-[ 4C)ANAm by using thin-layer chromatography in solvent system I. Symbols: El, total disintegrations per minute; (0, disintegrations per minute of 6-ANA; *, disintegrations per minute of 6-ANAm.
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ity originally present as 6-[14C]ANA in the medium was located inside the bacteria. These observations suggest that intracellular metabolites of 6-ANA may be formed during this period. These metabolites should be the direct precursors of 6-ANAm (see Fig. 1) and might also be responsible for the toxicity of 6-ANA. Intracellular metabolites of 6-ANA. To investigate the nature of the radioactive compound(s) that accumulates inside E. coli cells during incubation with 6[7-14C]ANA, a perchloric acid extract of these cells was prepared and subjected to Dowex-1 (formate) chromatography. Eight distinct radioactive peaks were obtained on chromatography of the perchloric acid-soluble material (Fig. 4). In this experiment as well as in the experiments shown in Fig. 5 and 6, 2 x 10-5 M PABA was added to the incubation medium to minimize the formation of any folic acid analogs of 6-ANA. In a control experiment (data not shown) in which PABA was omitted from the incubation medium, only one additional small radioactive peak was observed between peaks 7 and 8, and the relative sizes of peaks 1 through 8 were unchanged. It 9000-
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J. BACTERIOL.
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FIG. 5. Chromatography of metabolites labeled from 6-[7- "C]ANA and Na3nPO4. Incubation conditions were the same as described for Fig. 4, except that 0.6 mCi ofNa332PO4 was added to the incubation medium. After 6 h, cells were harvested by centrifugation, suspended in 2 ml of0.1 MHCIO4, and heated at 50°C for 5 min. After centrifugation the pellet was reextracted in the same manner with 1 ml of 0.1 M HClO,, and the combined supernatant solutions were neutralized and chromatographed ,as described for Fig. 4. (-) Disintegrations per minute of 14C, (-----) disintegrations per minute of 32p.
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FIIG. 4. Dowex-l (formate) chromatography of intracellular metabolite8 of 6-P14C]ANA. A 25-mi culture of Yates and Pardee minimal medium containing 0.5% glucose, 10-s M 6-[7-14CIANA (0.96 mCi/ mmol), and 2 x 10-5 M PABA was inoculated with 125 ml of a late-log-phase nutrient broth culture of W3899 NAm 11. After 6 h of incubation at 37°C and vigorous shakbing, the cells were harvested by centrifugation, suspended in 0.5 ml of distilled water, and disrupted 6068 for at 4C in a Branson Sonifier. An equal volume of15% perchloric acid was immediately added, and the precipitated protein was removed by centrifugation. The supernatant solution was neutralized with 10 N KOH and, after removal of the resulting KCIO4 by centrifugation, was applied to a 3- by 60-cm Dowex l-x8 formate column, which was eluted with water followed by a stepwise graldient of fotric acid as shown. Ten-milliliter fractions were collected, and 1-mi portions were counted in a 40% ethanoltoluene cocktail on a Packard Triotarb liquid scintillation spectrometer.
4 5 S
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FIG. 6. Chromatography of metabolites labeled from 6-[7-14CJANA and [8-3H]adenine. Experimental conditions were the same as described for Fig. 5, except that the incubation medium was reduced to 5 ml and also contained 10-4 M [8-3H]adenine (0.2 mCi). (-) Disintegrations per minute of 14C, (---) disintegrations per minute of 3H.
therefore appears that, under the conditions of our experiments, the major metabolites of 6ANA in E. coli are analogs of nicotinic acid derivatives rather than PABA derivatives. The major radioactive peak (peak 6) contained NAD, as shown by an alcohol dehydrogenase assay, and also comigrated with authentic NAD upon chromatography on a diethylaminoethyl-cellulose-bicarbonate column eluted with an NH4HCO gradient. A tentative identification of the other metabolites, based on their
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METABOLISM OF 6-AMINONICOTINIC ACID IN E. CoLI
relative mobilities as compared to literature reports (7, 15) of the mobilities of naturally occurring pyridine nucleotides and related compounds on similar Dowex-1 (formate) columns, is as follows: peak 1, 6-ANA riboside; peak 2, 6ANAm; peak 3, 6-ANMN; peak 4, 6-ANA; peak 5, unknown; peak 6, 6-ANAD plus NAD; peak 7, 6-ANaMN or 6-ANaAD; peak 8, 6-ANADP. Attempts at chemical characterization of these compounds met with difficulties because of the extremely small amounts available. We therefore approached this characterization by carrying out 6-[14C]ANA, 32P1, and [3H]adenine double-label experiments. The mono- and dinucleotide derivatives of 6-ANA should be labeled with both 14C and 32Pj; only the dinucleotide derivatives of 6-ANA should be labeled with both 14C and 3H, and the free bases, 6-ANA and 6-ANAm, and their mononucleosides should be labeled with 14C only. The results of these experiments are shown in Fig. 5 and 6. In both these experiments the harvested cells were extracted directly with perchloric acid rather than after sonic disruption to eliminate the possibility that some of the compounds seen in Fig. 4 could have been formed by enzymatic or chemical degradation during sonic treatment. In fact, peaks 4 and 5 appear to be significantly reduced in amount by this mode of extraction. However, all eight 14C peaks shown in Fig. 4 are also clearly visible in Fig. 5 and 6. Peak 8 is not shown in these figures since extremely high radioactivity from both 3H and 32P in this portion of the column eluate (presumably from adenine nucleotides and related compounds) makes any interpretation of double labeling difficult. In the 14C_32Pi experiment (Fig. 5) peaks 3, 5, 6, and 7 appeared to be labeled with both 14C and [32P]phosphate, suggesting that these compounds are mono- or dinucleotide derivatives of 6-ANA. The lack of exact correspondence between the 14C and [32P]phosphate peaks (especially noticeable in peak 6) is probably due to a partial resolution of 6-ANAD from NAD. NAD would contain some [32P]phosphate due to turnover processes (14), but, of course, no 14C In the 14C-3H experiment only peaks 6 and 7 showed significant 3H-labeling, indicating that they are dinucleotide derivatives of 6-ANA. The excess of 3H on the trailing edge of peak 6 may again be due to partial resolution of NAD and its 6-ANA analog. The small amount of tritium eluting with peak 1 is probably due to free adenine, which would be expected to appear in that area. The data obtained from these double-label experiments is consistent with the structure assignments of the radioactive peaks made on
793
the basis of relative elution position. The incorporation of [3H]adenine in peak 7 strongly suggests that this compound is 6-ANaAD rather than 6-ANaMN. The finding of [32P]phosphate but no 3H associated with peak 5 would suggest that this compound could be 6-ANaMN. However, nicotinic acid mononucleotide is more acidic than NAD and is eluted after NAD from Dowex-1 (formate) columns (15, 16). The identity of this minor peak remains an open question.
DISCUSSION The observation that an nadB pncA double mutant of E. coli excretes 6-[P4C]ANAm when incubated with 6-[7-14C]ANA supports the hypothesis that 6-ANA is a substrate for the enzymes of pyridine nucleotide metabolism in this organism and is converted to 6-ANAD by the same pathway and enzymes that form NAD from nicotinic acid. More direct evidence for this hypothesis is provided by the separation of the intracellular metabolites of 6-[7-14C]ANA and the tentative identification of some of these compounds as 6-ANA analogs of pyridine nucleotides. More than 75% of the total intracellular radioactivity arising from 6-[7-14C]ANA in the medium is present in a compound identified as 6-ANAD on the basis of its comigration with NAD on Dowex-1 (formate) and diethylaminoethyl-cellulose chromatography and incorporation into it of both 32P and [8-3H]adenine. The formation of 6-ANAD from 6-ANAm has been reported to occur in mammalian tissues via an exchange reaction with NAD catalyzed by NAD glycohydrolase (5). Since microbial NAD glycohydrolases are reported not to catalyze this exchange reaction (25) and since E. coli has been reported not to contain NAD glycohydrolase (2), it follows that 6-ANAD synthesis is probably catalyzed by the enzymes of the Preiss-Handler portion of the pyridine nucleotide cycle (6). Available evidence suggests that this pathway is not available for 6-ANAD synthesis in vertebrate cells since human and chicken erythrocytes form 6-ANaMN from 6ANA but do not further convert it to the next compound on the pathway, 6-ANaAD (4). The apparent discrepancy between the results of Wacker et al. (22), who reported finding only two radioactive metabolites of 6-[714C]ANA after incubation of E. stei with this compound, and our finding of at least seven pyridine nucleotide-related metabolites could be ascribed to differences in species or coiiditions. However, the discrepancy is more probably due to the superior resolving power of our separation methods and/or the greater sensitivity of our counting procedures.
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on the biosynthesis of nicotinamide adenine dinucleoThe biosynthesis of 6-ANAD from 6-ANA tide. III. Comparative in vivo studies on nicotinic could help explain the bacteriostatic effects of acid, nicotinamide, and quinolinic acid as precursors the latter compound. Unlike some other pyriof nicotinamide adenine dinucleotide. J. Biol. Chem. dine nucleotide analogs (12), 6-ANAD does not 241:3701-3707. function as an electron acceptor in enzymatic 8. I_sande, J., and A. B. Pardee. 1962. Regulation of pyridine nucleotide biosynthesis in Escherichia coli. oxidation-reduction reactions. According to J. Biol. Chem. 237:1305-1308. Pullman and Pullman (16), the lowest empty 9. Johnson, 0. H., D. E. Green, and R. Pauli. 1943. The electronic orbital is much higher in 6-ANAD antibacterial action of derivatives and analogues of paminobenzoic acid. J. Biol. Chem. 163:37-47. than in NAD. Therefore, the analog does not W. J. 1955. The inhibition of oulphanilamide have the pronounced electron acceptor proper- 10. Johnson, acetylation by aromatic and heterocyclic carboxamties of NAD and cannot function as an electron ides and carboxyhydrazides. Can. J. Biochem. Physcarrier in enzymatic oxidation-reduction reaciol. 33:107-111. tions. In addition, 6-ANAD and 6-ANADP have 11. Johnson, W. J., and J. D. McCall. 1955. 6-Aminonicotinamide - a potent nicotinamide antagonist. Science been shown to inhibit strongly several pyridine 122:834. nucleotide-linked dehydrogenases (5, 13). At 12. Kaplan, N. O., and M. M. Ciotti. 1956. Chemistry and least a portion of the bacteriostatic activity of properties of the 3-acetylpyridine analogue of diphosphopyridine nucleotide. J. Biol. Chem. 221:823-832. 6-ANA could, therefore, be explained by its enK., H. Kolbe, K. Keller, and H. Herken. 1970. zymatic conversion to 6-ANAD(P), which would 13. Lang, Der Kohlenhydratestoffwechsel des Gehirns nach both inhibit key dehydrogenase reactions and Blockade des Pentose-Phosphat-Weges durch 6-Amirepress the de novo biosynthesis of authentic nonicotinsiureamid Hoppe-Seyler's Z. Physiol. Chem. 361:1241-1252. NAD. However, formation of 6-ANA analog(s) P., and B. M. Olivera. 1973. of folic acid may also play a role in 6-ANA 14. Manlapa-Fernandez, Pyridine nucleotide metabolism in Escherichia coli. bacteriostasis since PABA has been reported to IV. Turnover. J. Biol. Chem. 248:5150-5155. antagonize the effects of 6-ANA in bacteria (9), 15. Preis, J., and P. Handier. 1958. Biosynthesis of diphos. phopyridine nucleotide. I. Identification of intermediand both we and Wacker et al. (22) have found ates. J. Biol. Chem. 233:488-492. that bacteria incorporate 6-[1"C]ANA into a 14C- 16. Pullman, B., and A. Pullman. 1959. On the 6-aminoniis whose fornation labeled compound supcotinamide antagonisms of DPN-dependent enzymatic systems. Cancer Res. 19:337-338. pressed by added PABA.
ACKNOWLEDGMENTS We wish to thank Alan Katz for his extraordinarily competent technical atance in this work. This research was supported by National Science Foundation grant no. PCM 74-20445 AO0. LITERATURE CITED 1. Andreoli, A. J., T. Grover, R. K. Gholson, and T. S. Matney. 1969. Evidence for a functional pyridine nucleotide cycle in Eacherichia coli. Biochim. Biophys. Acta 192:539-541. 2. Andreoll, A. J., T. W. Okita, R. Bloom, and T. A. Grover. 1972. The pyridine nucleotide cycle: presence of a nicotinamide mononucleotide-specific glycohydrolase in Escherichia coli. Biochem. Biophys. Res.
Commun. 49:264-269. 3. Chamberlain, J. G. 1967. Effects of acute vitamin replacement therapy on 6-aminonicotinamide induced cleft palate late in rat pregnancy. Proc. Soc. Exp. Biol. Med. 12:888-890. 4. Dietrich, L. S., and I. M. Friedland. 1960. -Aminonicotinamide and 6-aminonicotinic acid metabolism in nucleated and non-nucleated erythrocytes. Arch. Biochem. Biophys. 88:313-317. 5. Dietrich, L. S., I. M. Friedland, and L. A. Kaplan. 1958. Pyridine nucleotide metabolism: mechanism of action of the niacin antagonist, 6-aminonicotinamide. J. Biol. Chem. 283:964-968. 6. Gholon, R. L 1966. The pyridine nucleotide cycle. Nature (London) 212:933-935. 7. IJichi, H., A. Ichlyama, and 0. Hayahhi. 1966. Studies
17. Rath, C., and G. Prange. 1928. Ober eine neue Synthese der 2-Amino-nicotinsaure and Ihr Verhalten gegen Saltpertsaure. Justus Liebigs Ann. Chem. 467:1-10. 18. Shapino, D. M., L. S. Dietrich, and M. E. Shils. 1957. Quantitative biochemical differences between tumor and host as a basis for cancer chemotherapy. V. Niacin and 6-aminonicotinamide. Cancer Res. 17:600604. 19. Sundaram, T. K. 1967. Biosynthesis of nicotinamideadenine dinucleotide in Eacherichia coli. Biochim. Biophys. Acta 136:586-588. 20. Suzuki, N., J. Carlson, G. Griffith, and R. K. Gholsn. 1973. Studies on the de novo biosynthesis of NAD in Ewcherichia coli. V. Properties of the quinolinic acid synthetase system. Biochim. Biophys. Acta 304:309315. 21. Tritz, G. J., T. S. Matney, and R. K. Gholson. 1970. Mapping of the nadB locus adjacent to a previously undescribed purine locus in Eacherichia coli K-12. J. Bacteriol. 102:377-381. 22. Wacker, V. A., E. R. Zachmann, and L. Trager. 1967. Biosynthese FolsAure-und NAD-analoguer Verbindungen. Hoppe-Seyler's. Z. Physiol. Chem. 348:455459. 23. Wooley, J. G., M. K. Murphy, H. W. Bond, and T. D. Perrine. 1952. The effect of certain chemical compounds on the multiplication of T2 bacteriophage. J. Immunol. 68:523-530. 24. Yates, R. A., and A. B. Pardee. 1956. Pyridine biosynthesis inEwcherichia coli. J. Biol. Chem. 221:743-756. 25. Zatman, L. J., N. V. Kaplan, and S. P. Colowick. 1953. Inhibition of spleen diphosphopyridine nucleotidase by nicotinamide, an exchange reaction. J. Biol. Chem. 200:197-212.
JouRNAL oF BACTRIOLOGY, Sept. 1977, p. 789-794 Copyright 0 1977 American Society for Microbiology
Metabolism of 6-Aminonicotinic Acid in Escherichia colil JOHN R. COBB, SUSAN C. PEARCY, AND R. K. GHOLSON* Department ofBiochemistry, Oklahoma State University, Stillwater, Oklahoma 74074 Received for publication 4 March 1977
A late-log-phase culture of an Escherichia coli nadB pncA double mutant took up 6-[7-"4C]aminonicotinic acid and excreted 6-[14C]aminonicotinamide. This mutant also accumulated intracellularly several radioactive compounds which have been tentatively identified as 6-amino analogs of compounds in the pyridine nucleotide cycle. It is concluded that 6-aminonicotinamide and 6aminonicotinic acid probably exert at least a portion of their bacteriostatic effects by being metabolized, by the enzymes of the pyridine nucleotide cycle, to 6-aminonicotinamide adenine dinucleotide and 6-aminonicotinamide adenine dinucleotide phosphate. These compounds are not electron acceptors and are known inhibitors of some pyridine nucleotide-linked dehydrogenases.
6-Aminonicotinamide (6-ANAm) and 6-aminonicotinic acid (6-ANA) are analogs of nicotinamide and nicotinic acid, respectively. 6-ANA is also a structural analog and antimetabolite ofp-aminobenzoic acid (PABA) (9). The bacteriostasis produced by 6-ANA in Escherichia coli and Streptococcus haemolyticus was reversed by PABA (9). 6-ANA also inhibited multiplication of T2 bacteriophage, with the inhibition being reversed by nicotinamide, PABA, or acetate (23). 6-ANAm was also found to be toxic to mammals, with low doses causing blindness and paralysis in the extremities of rabbits and rats (10). The toxicity of 6-ANAm was reversed by nicotinamide, and it was concluded that 6ANAm is a potent antimetabolite of nicotinamide (11). In rats, 6-ANAm also has a teratogenetic effect, which is prevented by nicotinamide (3). In addition, 6-ANAm has been shown to have strong carcinostatic activity, which is reversed by nicotinamide (18). 6-ANAm was postulated to produce these effects in mammalian systems by conversion to an enzymatically inactive nicotinamide adenine dinucleotide (NAD) analog (11). Dietrich et al. (5) demonstrated the formation of 6-aminonicotinamide adenine dinucleotide (6-ANAD) and 6-aminonicotinamide adenine dinucleotide phosphate (6ANADP) from 6-ANAm in vivo by mouse tissues and in vitro by the exchange reaction catalyzed by pig brain NAD glycohydrolase. 6ANAD was found to inhibit several NADlinked dehydrogenases (5), and 6-ANADP was shown to be a potent inhibitor of 6-phosphogluconate dehydrogenase (13). Since 6-ANAm is 15 times more toxic than 6-ANA in mammals (W. I Journal article J-3291 of the Oklahoma State Agricultural Experiment Station.
789
J. Johnson and J. D. McCall, Fed. Proc., p. 284, 1956), the major pathway for the fonnation of the NAD and NAD phosphate (NADP) analogs in vivo is probably by a direct exchange of 6ANAm into NAD and NADP catalyzed by NAD glycohydrolase. Human and chicken erythrocytes form small quantities of 6-ANAm mononucleotide (6-ANMN) and 6-ANAD from 6ANAm, whereas 6-ANA mononucleotide (6ANaMN), but not 6-ANA adenine dinucleotide (6-ANaAD) or 6-ANAD, is formed from 6-ANA
(4).
The metabolic fate of 6-ANA in bacteria is less well documented. Wacker et al. (22) reported that 6-44C]ANA is incorporated into two radioactive compounds by Enterococcus stei. One was presumed to be a folic acid analog, since its formation was suppressed by addition of PABA to the culture medium, and the other was presumed to be an NAD analog because its formation was suppressed by nicotinic acid, it had the same Rf as NAD in the paper chromatography system used, and it was cleaved to 6ANA by perchloric acid treatment (22). This paper concerns the metabolic fate ofbacteriostatic concentrations of 6-ANA in E. coli. Since a thorough study has failed to show any NAD glycohydrolase activity in E. coli (2), our working hypothesis was that 6-ANAm and 6ANA are metabolized to NAD and NADP analogs by the enzymes of the pyridine nucleotide cycle (6, 14; see Fig. 1). The evidence obtained supports this hypothesis. MATERIALS AND METHODS Bacterial strains. The strains used in this study are niacin-requiring mutants of E. coli K-12 originally obtained from T. K. Sundaram (19) and designated by him W3899 and W3899 NAm 11. W3899 is
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blocked in the de novo pathway of NAD biosynthesis (nadB) (21), and W3899 NAm 11 contains an additional block in the conversion of nicotinamide to nicotinic acid (nadB pncA). Media and growth conditions. Cultures were grown with vigorous shaking or aeration at 37°C in the minimal medium of Yates and Pardee (24) with the additions noted in the tables and figure legends. For labeling experiments bacteria were grown in volumes of 5, 25, or 100 ml. Each culture was inoculated with a log-phase starter culture of 5% of its volume. The bacteria were harvested by centrifugation in a Sorvall RC2-B at 15,000 rpm for 15 min at 4°C in an 58-34 rotor. Chemicals. 6-ANAm was purchased from Aldrich Chemical Co. 6-ANA was prepared by refluxing 6ANAm in 50 weight volumes of 1.0 N NaOH for 1 h. The cooled solution was adjusted to pH 4 with glacial acetic acid, and the white precipitate of 6-ANA was collected and recrystallized from 40% acetic acid. The melting point was 297°C, in agreement with literature values. The product appeared homogeneous by thin-layer chromatography. Potassium [14C]cyanide, Hu2PO4, and [7-14C]nicotinic acid were purchased from New England Nuclear. [83H]adenine was obtained from Amersham/Searle. Nicotinic acid, nicotinamide, and alcohol dehydrogenase were purchased from the Sigma Chemical Co. PABA was purchased from Eastman Organic Chemicals; 2-amino-5-nitro-pyridine was from Aldrich Chemical Co., and 2,5-diphenyloxazole and 1,4bis-(6-phenyloxazolyl)-benzene were from the Packard Instrument Co. All other chemicals were obtained from local supply houses. The synthesis of 6-[7-'4C]ANA was based on a method described by Rath and Prange (17). This synthesis is similar to that described by Wacker et al. (22). 2,5-Diaminopyridine was synthesized from 2-amino-5-nitropyridine according to Rath and Prange (17), and 150 mg was dissolved in 1 ml of 0.8 N HCI in a 10-ml beaker. This solution was diazotized by the addition of 60 mg of NaNO2 dissolved in 0.5 ml of water. The solution was neutralized with solid NaCO, until CO2 evolution stopped and added to a solution of 19 mg of CuCN and 39 mg ofK14CN (1 mCi) in 2 ml of water in a 20-ml beaker. The mixture was heated for 1 h at 40°C. The reaction mixture was then evaporated carefully to dryness on a hot plate, and the solid was transferred to a Soxhlet cup and extracted with ether in a Soxhlet apparatus until no more yellow color was extracted. The ether was evaporated and the yellow solid 2-amino-5-[14Ccyanopyridine was hydrolyzed with 2 ml of 2 N NaOH at 100°C for 1 h. The solution was neutralized with HCI and applied to a Dowex-1 (formate) column (1 by 10 cm), which was washed first with 60 ml of water, which removed a radioactive peak (presumably nitrile), then with 60 ml of 10-4 M formic acid, which eluted a yellow compound, and then with 30 ml of 5 x 10-4 M formic acid. The 6-[7-14C]ANA was then eluted with 2 x 10-3 M formic acid. The fractions containing 6-[4C]ANA were pooled and lyophilized. The yield was 9.4 mg, and the specific activity was 0.96 mCi/mmol. 6-[7-14C]ANAm was prepared by heating the 2-amino-5-[14C]cyanopyridine in 2 ml
of water with Dowex-50 H+ at 100°C for 1 h. 6[U4C]ANAm was removed from the resin with water and purified on a Dowex-1 (formate) column. The radiochemical purity of the 6-[14C]ANA and 6['4C]ANAm was checked by radioautography after thin-layer chromatography in solvent systems I and II (see below). Several experiments to be described were performed with another preparation of 6-[714ClANA of slightly higher specific radioactivity. Thin-layer chromatography. Thin-layer chromatography was carried out with Analtech glass plates coated with cellulose 250 ,um thick containing fluorescent indicator. Solvent system I was n-butanol saturated with water-NH4OH, 66:6. Solvent system II was n-butanol saturated with water-glacial acetic acid, 66:3. Liquid scintiUation counting. Radioactivity was determined with a Packard Tri-Carb liquid scintillation spectrometer. The scintillation liquid was composed of 4 g of 2,5-diphenyloxazole, 0.2 g of 1,4bis-(5-phenyloxazolyl)-benzene, 400 ml of ethanol, and 600 ml of toluene per liter. The disintegrations per minute of each sample was calculated by the channels ratio method for quench correction and counting efficiency.
RESULTS A preliminary experiment was carried out to determine the gross metabolic fate of 6[14C]ANA in E. coli. Since NAD synthesis in E. coli can occur either by a de novo pathway from aspartate (20) or by utilization of the exogenously supplied base, an nadB mutant, which lacks the former pathway, was used to increase the utilization of the exogenously supplied analog. This strain (W3899 NAm 11) also carries a mutation at the pncA locus, which is the structural gene for nicotinamide deamidase (19). This block in the pyridine nucleotide cycle prevents utilization of nicotinamide; hence, any nicotinamide generated during normal operation of the pyridine nucleotide cycle will not be further metabolized and will accumulate in the medium (Fig. 1). It has been previously shown that this double mutant excretes [14C]nicotinamide into the medium when incubated in the presence of [14C]nicotinic acid (1). Since the only known reaction sequence for this conversion is the amidation of nicotinic acid adenine dinucleotide to NAD and the subsequent degradation of NAD to nicotinamide, the excretion of nicotinamide was considered strong evidence for the presence of a functioning pyridine nucleotide cycle in E. coli (1, 14). When washed cells of W3899 NAm 11 were incubated with 6-[7-14C]ANA for 6 h, two major compounds were recovered from the incubation medium (Fig. 2). The compound eluted from Dowex-1 (formate) with 1 N formic acid (peak II) was identified as unchanged 6-[14C]ANA by thin-layer chromatography in solvent systems I
METABOLISM OF 6-AMINONICOTINIC ACID IN E. COLI
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starting material was found in the medium. When W3899 (nadB) was incubated with these "C-labeled amides, the corresponding acids NA 4-- NAm-NMN were excreted into the medium. These results ACID pnc A establish that 6-ANAm is a substrate for nicoFIG. 1. Pyridine nucleotide cycle in E. coli. nadA, tinamide deamidase. nadB, nadC, and pncA show the metabolic location The deamidation of these amides to the correof known genetic lesions. Abbreviations: DHAP, di- sponding acids, which accumulate in the mehydroxyacetone phosphate; NaMN, nicotinic acid dium of E. coli W3899 (which has an intact mononucleotide; NaAD, nicotinic acid adenine dinunucleotide cycle), suggests that nicocleotide; NMN, nicotinamide mononucleotide; NAm, pyridine tinamide deamidase is unregulated and supnicotinamide; NA, nicotinic acid. ports the conclusion of Imsande and Pardee (8) that the formation of nicotinic acid mononucleotide from nicotinic acid is a rate-limiting 1.0 step in NAD biosynthesis in E. coli. H20 I N Formate , Time course of 6-414CIANA uptake and 6o.s [14C]ANAm excretion' When a culture of E. 0.8 II coli W3899 NAm 11 containing approximately 2 0.7 x 10'1 cells/ml was incubated in minimal me1I 0.6 dium with 2 x 1O-5 M 6-[7-14C]ANA, this com, 1 0.5 pound was taken up continuously by the bacte~~~~~~~~~~~~~~~~~~~~I 0.4 ria for the 6-h duration of the experiment (Fig. A------tI 3). At 90 min after the beginning of incubation 0.3 6-44C]ANAm was detectable in the medium, 0.2 I~~~~~~~ and the amount of this compound present con0.1 tinued to increase linearly for the rest ofthe 6-h experiment. After an initial period the amount 5 I5 25 35 45 55 of total extracellular radioactivity decreased Fraction No. FIG. 2. Dowex-1 (formate) chromatography ofme- continuously over the course ofthe experiment. dium from an E. coli nadB pncA double mutant After 6 h approximately 45% of the radioactivincubated with 6-['4C]ANA. A late-log-phase culture CO
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ofE. coli W3899 NAm 11 was washed in 0.9% saline and suspended at a concentration of approximately 3 x 109 cellslml in 10 ml of minimal medium containing 2 x 10-5 M 6-[14C]ANA (3.2 mCi/mmol) and shaken at 37°C for 6 h. The cells were removed by centrifugation, 1 p,mol each of 6-ANA and 6-ANAm were added, and the medium was placed on a 1- by 35-cm column of Dowex 1-x8 formate, which was eluted with water and 1 N formic acid. Fractions of 4 ml were collected, and absorbance at 260 nm (A2nd) and radioactivity were determined.
15 13 11I
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9
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and II. The compound eluted with water (peak I) was identified as 6-['4C]ANAm by chromatography in these solvent systems and by hydrolysis to 6-14C]ANA in 1 N NaOH. This conversion of 6-ANA to 6-ANAm by W3899 NAm 11 provides strong presumptive evidence that the 6-ANA is converted to pyridine nucleotide analogs and then to 6-ANAm by the reactions of the pyridine nucleotide cycle. Control experiments were carried out to show that the pncA mutant did not excrete "4C-labeled compounds upon incubation with 6-[74CIANAm or [7-14C]nicotinamide. When W3899 NAm 11 cells (nadB pncA) were incubated with 6-[14C]ANAm or [14C]nicotinamide under the conditions described above, only the
30
90 120
180 240 300 360 Time (min)
FIG. 3. Uptake of 6-[14C]ANA and excretion of 6['4C]ANAm by an nadB pncA mutant as a function of time. A 100-ml amount of medium containing 2 x 10-5 M 6-[7-14CIANA (32 mCilmmol) was inoculated with approximately 2 x 1011 cells harvested
from a late-log-phase culture of E. coli W3899 NAm 11. One-milliliter samples were removed at the times indicated, and the bacteria were separated by centrifugation. The supernatant solutions were analyzed for 6-P14CJANA and 6-[ 4C)ANAm by using thin-layer chromatography in solvent system I. Symbols: El, total disintegrations per minute; (0, disintegrations per minute of 6-ANA; *, disintegrations per minute of 6-ANAm.
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ity originally present as 6-[14C]ANA in the medium was located inside the bacteria. These observations suggest that intracellular metabolites of 6-ANA may be formed during this period. These metabolites should be the direct precursors of 6-ANAm (see Fig. 1) and might also be responsible for the toxicity of 6-ANA. Intracellular metabolites of 6-ANA. To investigate the nature of the radioactive compound(s) that accumulates inside E. coli cells during incubation with 6[7-14C]ANA, a perchloric acid extract of these cells was prepared and subjected to Dowex-1 (formate) chromatography. Eight distinct radioactive peaks were obtained on chromatography of the perchloric acid-soluble material (Fig. 4). In this experiment as well as in the experiments shown in Fig. 5 and 6, 2 x 10-5 M PABA was added to the incubation medium to minimize the formation of any folic acid analogs of 6-ANA. In a control experiment (data not shown) in which PABA was omitted from the incubation medium, only one additional small radioactive peak was observed between peaks 7 and 8, and the relative sizes of peaks 1 through 8 were unchanged. It 9000-
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FIG. 5. Chromatography of metabolites labeled from 6-[7- "C]ANA and Na3nPO4. Incubation conditions were the same as described for Fig. 4, except that 0.6 mCi ofNa332PO4 was added to the incubation medium. After 6 h, cells were harvested by centrifugation, suspended in 2 ml of0.1 MHCIO4, and heated at 50°C for 5 min. After centrifugation the pellet was reextracted in the same manner with 1 ml of 0.1 M HClO,, and the combined supernatant solutions were neutralized and chromatographed ,as described for Fig. 4. (-) Disintegrations per minute of 14C, (-----) disintegrations per minute of 32p.
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FIIG. 4. Dowex-l (formate) chromatography of intracellular metabolite8 of 6-P14C]ANA. A 25-mi culture of Yates and Pardee minimal medium containing 0.5% glucose, 10-s M 6-[7-14CIANA (0.96 mCi/ mmol), and 2 x 10-5 M PABA was inoculated with 125 ml of a late-log-phase nutrient broth culture of W3899 NAm 11. After 6 h of incubation at 37°C and vigorous shakbing, the cells were harvested by centrifugation, suspended in 0.5 ml of distilled water, and disrupted 6068 for at 4C in a Branson Sonifier. An equal volume of15% perchloric acid was immediately added, and the precipitated protein was removed by centrifugation. The supernatant solution was neutralized with 10 N KOH and, after removal of the resulting KCIO4 by centrifugation, was applied to a 3- by 60-cm Dowex l-x8 formate column, which was eluted with water followed by a stepwise graldient of fotric acid as shown. Ten-milliliter fractions were collected, and 1-mi portions were counted in a 40% ethanoltoluene cocktail on a Packard Triotarb liquid scintillation spectrometer.
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FIG. 6. Chromatography of metabolites labeled from 6-[7-14CJANA and [8-3H]adenine. Experimental conditions were the same as described for Fig. 5, except that the incubation medium was reduced to 5 ml and also contained 10-4 M [8-3H]adenine (0.2 mCi). (-) Disintegrations per minute of 14C, (---) disintegrations per minute of 3H.
therefore appears that, under the conditions of our experiments, the major metabolites of 6ANA in E. coli are analogs of nicotinic acid derivatives rather than PABA derivatives. The major radioactive peak (peak 6) contained NAD, as shown by an alcohol dehydrogenase assay, and also comigrated with authentic NAD upon chromatography on a diethylaminoethyl-cellulose-bicarbonate column eluted with an NH4HCO gradient. A tentative identification of the other metabolites, based on their
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relative mobilities as compared to literature reports (7, 15) of the mobilities of naturally occurring pyridine nucleotides and related compounds on similar Dowex-1 (formate) columns, is as follows: peak 1, 6-ANA riboside; peak 2, 6ANAm; peak 3, 6-ANMN; peak 4, 6-ANA; peak 5, unknown; peak 6, 6-ANAD plus NAD; peak 7, 6-ANaMN or 6-ANaAD; peak 8, 6-ANADP. Attempts at chemical characterization of these compounds met with difficulties because of the extremely small amounts available. We therefore approached this characterization by carrying out 6-[14C]ANA, 32P1, and [3H]adenine double-label experiments. The mono- and dinucleotide derivatives of 6-ANA should be labeled with both 14C and 32Pj; only the dinucleotide derivatives of 6-ANA should be labeled with both 14C and 3H, and the free bases, 6-ANA and 6-ANAm, and their mononucleosides should be labeled with 14C only. The results of these experiments are shown in Fig. 5 and 6. In both these experiments the harvested cells were extracted directly with perchloric acid rather than after sonic disruption to eliminate the possibility that some of the compounds seen in Fig. 4 could have been formed by enzymatic or chemical degradation during sonic treatment. In fact, peaks 4 and 5 appear to be significantly reduced in amount by this mode of extraction. However, all eight 14C peaks shown in Fig. 4 are also clearly visible in Fig. 5 and 6. Peak 8 is not shown in these figures since extremely high radioactivity from both 3H and 32P in this portion of the column eluate (presumably from adenine nucleotides and related compounds) makes any interpretation of double labeling difficult. In the 14C_32Pi experiment (Fig. 5) peaks 3, 5, 6, and 7 appeared to be labeled with both 14C and [32P]phosphate, suggesting that these compounds are mono- or dinucleotide derivatives of 6-ANA. The lack of exact correspondence between the 14C and [32P]phosphate peaks (especially noticeable in peak 6) is probably due to a partial resolution of 6-ANAD from NAD. NAD would contain some [32P]phosphate due to turnover processes (14), but, of course, no 14C In the 14C-3H experiment only peaks 6 and 7 showed significant 3H-labeling, indicating that they are dinucleotide derivatives of 6-ANA. The excess of 3H on the trailing edge of peak 6 may again be due to partial resolution of NAD and its 6-ANA analog. The small amount of tritium eluting with peak 1 is probably due to free adenine, which would be expected to appear in that area. The data obtained from these double-label experiments is consistent with the structure assignments of the radioactive peaks made on
793
the basis of relative elution position. The incorporation of [3H]adenine in peak 7 strongly suggests that this compound is 6-ANaAD rather than 6-ANaMN. The finding of [32P]phosphate but no 3H associated with peak 5 would suggest that this compound could be 6-ANaMN. However, nicotinic acid mononucleotide is more acidic than NAD and is eluted after NAD from Dowex-1 (formate) columns (15, 16). The identity of this minor peak remains an open question.
DISCUSSION The observation that an nadB pncA double mutant of E. coli excretes 6-[P4C]ANAm when incubated with 6-[7-14C]ANA supports the hypothesis that 6-ANA is a substrate for the enzymes of pyridine nucleotide metabolism in this organism and is converted to 6-ANAD by the same pathway and enzymes that form NAD from nicotinic acid. More direct evidence for this hypothesis is provided by the separation of the intracellular metabolites of 6-[7-14C]ANA and the tentative identification of some of these compounds as 6-ANA analogs of pyridine nucleotides. More than 75% of the total intracellular radioactivity arising from 6-[7-14C]ANA in the medium is present in a compound identified as 6-ANAD on the basis of its comigration with NAD on Dowex-1 (formate) and diethylaminoethyl-cellulose chromatography and incorporation into it of both 32P and [8-3H]adenine. The formation of 6-ANAD from 6-ANAm has been reported to occur in mammalian tissues via an exchange reaction with NAD catalyzed by NAD glycohydrolase (5). Since microbial NAD glycohydrolases are reported not to catalyze this exchange reaction (25) and since E. coli has been reported not to contain NAD glycohydrolase (2), it follows that 6-ANAD synthesis is probably catalyzed by the enzymes of the Preiss-Handler portion of the pyridine nucleotide cycle (6). Available evidence suggests that this pathway is not available for 6-ANAD synthesis in vertebrate cells since human and chicken erythrocytes form 6-ANaMN from 6ANA but do not further convert it to the next compound on the pathway, 6-ANaAD (4). The apparent discrepancy between the results of Wacker et al. (22), who reported finding only two radioactive metabolites of 6-[714C]ANA after incubation of E. stei with this compound, and our finding of at least seven pyridine nucleotide-related metabolites could be ascribed to differences in species or coiiditions. However, the discrepancy is more probably due to the superior resolving power of our separation methods and/or the greater sensitivity of our counting procedures.
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on the biosynthesis of nicotinamide adenine dinucleoThe biosynthesis of 6-ANAD from 6-ANA tide. III. Comparative in vivo studies on nicotinic could help explain the bacteriostatic effects of acid, nicotinamide, and quinolinic acid as precursors the latter compound. Unlike some other pyriof nicotinamide adenine dinucleotide. J. Biol. Chem. dine nucleotide analogs (12), 6-ANAD does not 241:3701-3707. function as an electron acceptor in enzymatic 8. I_sande, J., and A. B. Pardee. 1962. Regulation of pyridine nucleotide biosynthesis in Escherichia coli. oxidation-reduction reactions. According to J. Biol. Chem. 237:1305-1308. Pullman and Pullman (16), the lowest empty 9. Johnson, 0. H., D. E. Green, and R. Pauli. 1943. The electronic orbital is much higher in 6-ANAD antibacterial action of derivatives and analogues of paminobenzoic acid. J. Biol. Chem. 163:37-47. than in NAD. Therefore, the analog does not W. J. 1955. The inhibition of oulphanilamide have the pronounced electron acceptor proper- 10. Johnson, acetylation by aromatic and heterocyclic carboxamties of NAD and cannot function as an electron ides and carboxyhydrazides. Can. J. Biochem. Physcarrier in enzymatic oxidation-reduction reaciol. 33:107-111. tions. In addition, 6-ANAD and 6-ANADP have 11. Johnson, W. J., and J. D. McCall. 1955. 6-Aminonicotinamide - a potent nicotinamide antagonist. Science been shown to inhibit strongly several pyridine 122:834. nucleotide-linked dehydrogenases (5, 13). At 12. Kaplan, N. O., and M. M. Ciotti. 1956. Chemistry and least a portion of the bacteriostatic activity of properties of the 3-acetylpyridine analogue of diphosphopyridine nucleotide. J. Biol. Chem. 221:823-832. 6-ANA could, therefore, be explained by its enK., H. Kolbe, K. Keller, and H. Herken. 1970. zymatic conversion to 6-ANAD(P), which would 13. Lang, Der Kohlenhydratestoffwechsel des Gehirns nach both inhibit key dehydrogenase reactions and Blockade des Pentose-Phosphat-Weges durch 6-Amirepress the de novo biosynthesis of authentic nonicotinsiureamid Hoppe-Seyler's Z. Physiol. Chem. 361:1241-1252. NAD. However, formation of 6-ANA analog(s) P., and B. M. Olivera. 1973. of folic acid may also play a role in 6-ANA 14. Manlapa-Fernandez, Pyridine nucleotide metabolism in Escherichia coli. bacteriostasis since PABA has been reported to IV. Turnover. J. Biol. Chem. 248:5150-5155. antagonize the effects of 6-ANA in bacteria (9), 15. Preis, J., and P. Handier. 1958. Biosynthesis of diphos. phopyridine nucleotide. I. Identification of intermediand both we and Wacker et al. (22) have found ates. J. Biol. Chem. 233:488-492. that bacteria incorporate 6-[1"C]ANA into a 14C- 16. Pullman, B., and A. Pullman. 1959. On the 6-aminoniis whose fornation labeled compound supcotinamide antagonisms of DPN-dependent enzymatic systems. Cancer Res. 19:337-338. pressed by added PABA.
ACKNOWLEDGMENTS We wish to thank Alan Katz for his extraordinarily competent technical atance in this work. This research was supported by National Science Foundation grant no. PCM 74-20445 AO0. LITERATURE CITED 1. Andreoli, A. J., T. Grover, R. K. Gholson, and T. S. Matney. 1969. Evidence for a functional pyridine nucleotide cycle in Eacherichia coli. Biochim. Biophys. Acta 192:539-541. 2. Andreoll, A. J., T. W. Okita, R. Bloom, and T. A. Grover. 1972. The pyridine nucleotide cycle: presence of a nicotinamide mononucleotide-specific glycohydrolase in Escherichia coli. Biochem. Biophys. Res.
Commun. 49:264-269. 3. Chamberlain, J. G. 1967. Effects of acute vitamin replacement therapy on 6-aminonicotinamide induced cleft palate late in rat pregnancy. Proc. Soc. Exp. Biol. Med. 12:888-890. 4. Dietrich, L. S., and I. M. Friedland. 1960. -Aminonicotinamide and 6-aminonicotinic acid metabolism in nucleated and non-nucleated erythrocytes. Arch. Biochem. Biophys. 88:313-317. 5. Dietrich, L. S., I. M. Friedland, and L. A. Kaplan. 1958. Pyridine nucleotide metabolism: mechanism of action of the niacin antagonist, 6-aminonicotinamide. J. Biol. Chem. 283:964-968. 6. Gholon, R. L 1966. The pyridine nucleotide cycle. Nature (London) 212:933-935. 7. IJichi, H., A. Ichlyama, and 0. Hayahhi. 1966. Studies
17. Rath, C., and G. Prange. 1928. Ober eine neue Synthese der 2-Amino-nicotinsaure and Ihr Verhalten gegen Saltpertsaure. Justus Liebigs Ann. Chem. 467:1-10. 18. Shapino, D. M., L. S. Dietrich, and M. E. Shils. 1957. Quantitative biochemical differences between tumor and host as a basis for cancer chemotherapy. V. Niacin and 6-aminonicotinamide. Cancer Res. 17:600604. 19. Sundaram, T. K. 1967. Biosynthesis of nicotinamideadenine dinucleotide in Eacherichia coli. Biochim. Biophys. Acta 136:586-588. 20. Suzuki, N., J. Carlson, G. Griffith, and R. K. Gholsn. 1973. Studies on the de novo biosynthesis of NAD in Ewcherichia coli. V. Properties of the quinolinic acid synthetase system. Biochim. Biophys. Acta 304:309315. 21. Tritz, G. J., T. S. Matney, and R. K. Gholson. 1970. Mapping of the nadB locus adjacent to a previously undescribed purine locus in Eacherichia coli K-12. J. Bacteriol. 102:377-381. 22. Wacker, V. A., E. R. Zachmann, and L. Trager. 1967. Biosynthese FolsAure-und NAD-analoguer Verbindungen. Hoppe-Seyler's. Z. Physiol. Chem. 348:455459. 23. Wooley, J. G., M. K. Murphy, H. W. Bond, and T. D. Perrine. 1952. The effect of certain chemical compounds on the multiplication of T2 bacteriophage. J. Immunol. 68:523-530. 24. Yates, R. A., and A. B. Pardee. 1956. Pyridine biosynthesis inEwcherichia coli. J. Biol. Chem. 221:743-756. 25. Zatman, L. J., N. V. Kaplan, and S. P. Colowick. 1953. Inhibition of spleen diphosphopyridine nucleotidase by nicotinamide, an exchange reaction. J. Biol. Chem. 200:197-212.
