Metabolism of n-propylamine, Isopropylamine, and

Transcription

1 JouRNAL of BACMIOLWGY, OCt. 1975, p Copyright 1975 American Society for Microbiology Vol. 124, No. 1 Printed in U.S.A. Metabolism of n-propylamine, Isopropylamine, and 1,3-Propane Diamine by Mycobacterium convolutum C. E. CERNIGLIA AND J. J. PERRY* Department of Microbiology, North Carolina State University, Raleigh, North Carolina 2767 Received for publication 23 June 1975 WMycobacterium convolutum strain NPA-1 can utilize n-propylamine (NPA), isopropylamine (IPA), and 1,3-propane diamine (PD) as sole source of carbon, nitrogen, and energy. Enzyme assays, fatty acid profiles, and "4CO incorporation experiments indicate that NPA is deaminated to propionate and further metabolized via the methylmalonyl succinate pathway, and [PA and PD were metabolized (after deamination) through a C, + C, cleavage. An inducible amine dehydrogenase was present in cell extracts after growth on the three amines. Polyacrylamide gel electrophoresis of cell extracts from NPA- and IPA-grown cells yielded one major band of amine dehydrogenase activity. When extracts of NPA-grown cells were assayed with NPA, [PA, or PD as substrate, the relative position of the major band on gel electrophoresis was equivalent. Similar results were obtained with extracts prepared from IPA-grown cells. Sephadex G-1 chromatography also indicated one major peak of activity. This suggests that one enzyme of broad specificity is involved in deamination of IPA, NPA, and PD. IPA-grown cells utilized NPA readily, whereas NPA-grown cells could not utilize [PA without lag. Since amine dehydrogenase activity was present in extracts of cells after growth on either substrate, this lag was probably due to the inability to transport IPA without an induction period. The molecular weight of the amine dehydrogenase was approximately 38,5 as determined by gel filtration. Recently we described the metabolic pathway involved in the assimilation of n-propylamine (NPA) by a microorganism originally isolated by enrichment on propane (2). This organism, designated Mycobacterium convolutum strain R22 (1), oxidized the primary amine to propionate and utilized the propionate thus formed via carboxylation to methylmalonate, isomerization to succinate, and further through the tricarboxylic acid cycle. This was in contrast to propane which was oxidized to acetol, cleaved to acetate and a 1-carbon compound, and further metabolized through the isocitrate lyase shunt. The ability to utilize NPA as sole carbon and energy source is widespread in stock culture orgainisms that are capable of growth on propane. However, none of these organisms that grew on NPA would utilize isopropylamine (IPA) as substrate but grew well on isopropanol and acetone (15). It has been reported by Eady and Large (7, 8) that the amine dehydrogenase of Pseudomonas AM1 has significant activity on primary amines, e.g. NPA, but little activity on isoamines. To learn more about the utilization of I Paper no of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, N. C alkyl-amines in bacteria, several organisms were isolated from soil that grew with NPA as sole source of carbon. One of these organisms, designated M. convolutum strain NPA-1, grew with NPA, IPA, and 1,3-propane diamine (PD) as sole source of carbon, nitrogen, and energy. This study is concerned with the metabolism of NPA, [PA, and PD by strain NPA-1 MATERIALS AND METHODS Microorganism. A short, gram-positive, non-acidfast rod was isolated by enrichment culture with NPA as substrate by methods previously described (15). It was identified as a strain of M. convolutum by Ruth E. Gordon, Rutgers University (personal communication). This organism can utilize, as sole source of carbon and energy, all n-alkanes from C, through C,, and a wide array of long-chain alkenes, ketones, fatty acids, NPA, IPA, and PD. Malonate will also serve as substrate for growth of this strain of M. convolutum. Media and growth conditions. M. convolutum was cultured on L-salts medium (13) supplemented with the appropriate carbon source. Isopropanol, n-propanol, NPA, IPA, PD, propionate, glucose, malonate, and acetate were added at a concentration of.2%. The amines were peutralized with HCl and filter sterilized. For growth on all substrates the inoculum was preadapted by growth on that substrate. All respiration studies were as previously described (2, 17). 285

2 286 CERNIGLIA AND PERRY Enzyme assays. Crude cell-free extracts for isocitrate lyase determination were prepared by suspending cells in tris(hydroxymethyl)aminomethane buffer (ph 7.9) and disrupting them in a French pressure cell. Extracts for amine dehydrogenase were made in 67 mm sodium phosphate buffer (ph 7.5). Cell debris was removed by centrifugation at 17, x g for 3 min at 4 C. The supernatant fluid was subjected to centrifugation at 1, x g for 6 min at 4 C. The supernatant was decanted and immediately assayed for enzyme activity. Protein was quantitated by the method of Lowry et al. (14). Isocitrate lyase (threo-d.- isocitrate glyoxylate lyase, EC ) and amine dehydrogenase were determined by methods previously described (2). Fatty acid analysis. Cellular fatty acids were converted to the corresponding methyl ester and identified as described (6, 18). The area of each chromatographic peak was determined by the method of Carroll (4). "CO2 incorporation experiments. The procedure of Smith and Kornberg (16) was utilized for "CO2 incorporation experiments, with modifications as reported (17). Gel filtration. A column of Sephadex G-1 (2.6 by 4 cm) was equilibrated at 4 C with 67 mm phosphate buffer at ph 7.5, and void volume of the column was determined with 1. ml of freshly prepared blue dextran 2 (1 mg/ml). The calibration curve was established with the purified protein standards. Two separate samples were employed, with aldolase and chymotrypsinogen A being applied to the column in one, followed by ovalbumin and ribonculease A in the second analysis. After standardization, the partially purified amine dehydrogenase was placed on the column, the elution volume was monitored by assaying for activity, and the molecular weight was estimated (19). Polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis was carried out by the method of Davis (5). Gels containing 7.5% acrylamide,.2% bis-acrylamide,.84% (vol/vol) N,N,N',N'-tetramethylethylenediamine and.175% (wt/vol) ammonium persulfate were employed. After setting of the gel, a 1% glycerol enzyme preparation containing 2 Ag of protein to which bromophenol blue dye had been added was transferred into the gel. Separation was obtained by using a tris(hydroxymethyl)aminomethane glycine buffer at ph 7.9 with a current of 1.5 ma per gel at 4 C until the dye had migrated nearly to the opposite end of the gel. Protein bands were detected by staining the gels with.25% Coomassie blue in 9.2% acetic acid-5% methanol. Enzyme activity in the gels was localized by coupling amine dehydrogenase activity to the reduction of tetrazolium chloride. The gels were removed from the tubes and incubated for 3 min in 5 ml of the following reaction mixture: 67 mm phosphate buffer (ph 7.5), 1. Mmol of phenazine methyl sulfate, 2Mmol of NPA or IPA (ph 7.), and.4 mg of tetrazolium chloride. RESULTS M. convolutum cells were harvested after growth on various substrates, and cell-free extracts were prepared and assayed for isocitrate lyase activity (Table 1). There was no detectable enzymatic activity in extracts of cells grown on NPA, propionate, or n-propanol. Growth on acetate, propane, malonate, IPA, or PD resulted in the induction of isocitrate lyase activity. Respirometer studies demonstrated that M. convolutum grown on IPA utilized NPA without lag but nonproliferating cells after growth on NPA had a 2- to 3-min lag period before utilizing IPA. Nonproliferating M. convolutum cells that were grown on acetate, propionate, NPA, IPA, and PD were exposed to the corresponding growth substrate in the presence of NaH 14CO,. The amount of radioactivity recovered in pyruvate during metabolism of these substrates is shown in Table 2. Significant radiolabeled pyruvate was produced when NPA and propionate served as substrate and little was evident in pyruvate produced by cells metabolizing IPA and PD. The fatty acids present in M. convolutum after growth on acetate, propionate, malonate, IPA, NPA, isopropanol, or PD were subjected to chromatographic analysis (Table 3). The total percentage of odd-numbered normal fatty acids (C 15, C 17, C 17: 1) was highest in the cells grown on propionate and NPA. A predominance of even carbon fatty acids was obtained when this organism was grown on acetate, isopropanol, malonate, IPA, and PD. The level of amine dehydrogenase activity in cells after growth on NPA, IPA, and PD is shown in Table 4. No detectable amine dehydrogenase activity was evident in crude extracts from glucose or propionate grown cells. Further study on the amine dehydrogenase activity in NPA, IPA, and PD cell-free preparations was conducted after high-speed centrifugation at 1, x g for 9 min. Examination of the TABLE 1. Level of isocitrate lyase in cell extracts of M. convolutum strain NPA-1 after growth on various substrates Growth substrate Sp acta Acetate 1.2 n-propanol Propionate Propane.6 NPA IPA 1.6 PD 1.3 Malonate.9 J. BACTERIOL. a Units per milligram of protein in which 1 U is the amount of enzyme necessary for the cleavage of 1 Mmol of isocitrate in 1 min at 3 C.

3 VOL. 124, 1975 AMINE METABOLISM IN M. CONVOLUTUM 287 TABLE 2. Relative amount of [14C ]carbon dioxide incorporated into pyruvate produced by nonproliferating cells of M. convolutum strain NPA-1 during oxidation of malate, acetate, NPA, propionate, IPA and PDa Substrates Pyruvate from cells grown on (counts/min/mg)b Acetate Propionate NPA IPA PD Malate Malate + NaAsO, Acetate 2 Acetate + NaAsO, 28 NPA 52 NPA + NaAsO, 1,4 IPA 3 IPA + NaAsO, 19 PD 6 PD + NaAsO, 4 Propionate 18 Propionate + NaAsO, 1,242 a Each vessel contained 5 jsmol of substrate and 1 MCi of NaH "CO,; 4 /Amol of NaAsO, was added. Cells were suspended in.1 M tris(hydroxymethyl)aminomethane buffer (ph 7.9). Final volume was 3.5 ml and incubation was at 3 C for 15 min. bvalues corrected for background and endogenous counts. TABLE 3. Fatty acid Fatty acid composition of M. convolutum strain NPA-1 after growth on various substrates Growth substrate Acetate Propionate. Isopropanol IPA NPA PD Malonate C 12 Tra Tr Tr Tr Tr Tr Tr C 13 Tr Tr Tr Tr 1.6 Tr Tr C14 2.1b C1s Tr 1.5 Cis C ND ND C 17 NDa 7.9 ND ND C 17: Br C 17c Tr ND 4.5 ND ND ND ND C1s ND ND ND ND ND ND 3.4 CI: ND ND Br C1,c Tr ND Br C, ND ND 24.8 Tr Odd Even atr, Trace; ND, none detected. b Recorded as percentage of total fatty acids present. c Br C 17 and Br C,8 = 1 methyl branched. sedimented pellet and supernatant solution revealed that the amine dehydrogenase activity remained in the supernatant. Polyacrylamide gel electrophoresis of cell extracts of NPAand IPA-grown cells yielded one major band of enzyme activity (Fig. 1). Filtration of crude extracts of NPA and IPA grown cells through G-1 Sephadex gel resulted in the elution of the amine dehydrogenase in one activity peak fraction (Fig. 2). This partially purified amine dehydrogenase was used for molecular weight estimation with a precalibrated G-1 Sephadex column. A calibration curve was drawn (Fig. 3) and the resulting enzyme activity peak gave a Kavg value corresponding to a molecular weight of approximately 38,5. DISCUSSION The pathways by which propane, acetone, propionate, and NPA were metabolized by selected hydrocarbon-utilizing microorganisms has been outlined in recent reports (2, 18). Propane is oxidized to acetone and further to acetol which is cleaved to acetate and an unidentified one-carbon compound; propionate is metabolized via a carboxylation to methylmalonate and isomerized to succinate. The

4 288 CERNIGLIA AND PERRY TABLE 4. Level of amine dehydrogenase activity in M. convolutum strain NPA-1 crude extract and in the supernatant fraction and pellet after high-speed centrifugationa Determinants Growth substrate Specific activity on: NPA IPA J. BACTERIOL. indicate that, in both organisms, terminally oxidized intermediates (propionaldehyde, propionate) are involved in the utilization of NPA. The absence of detectable isocitrate lyase activity (Table 1) suggests that there is no significant accumulation of 2-carbon intermediates formed Crude extract Glucose Propionate NPA IPA PD Supernatant fraction NPA from 1, x g IPA for 9 min PD Pellet from 1, NPA x gfor9min IPA athe organisms was grown on glucose, propionate, NPA, IPA, and PD. Units per milligram of protein in which 1 U is the amount of enzyme necessary to reduce 1 gmol of dichlorophenolindophenol per min at 28 C. NPA I PA rk ^ A N PA I PA FIG. 1. Polyacrylamide gel migration of the amine dehydrogenase from M. convolutum strain NPA-1 after growth on NPA and IPA. Gel A contained extract from NPA-grown cells with NPA and IPA as substrate and gel B contained extract from IPA-grown cells on the same substrates. -, Amine dehydrogenase. major pathway for propane utilization would therefore be the isocitrate lyase shunt and for propionate, the tricarboxylic acid cycle. Results were also presented (2) suggesting that, in M. convolutum strain R-22, NPA was deaminated to propionate and utilized via the methylmalonate pathway. A comparison of the results obtained with M. convolutum strain R-22 with those of M. convolutum strain NPA-1 after growth on NPA B i z 4 4 FRACTION NUMBER FIG. 2. Gel filtration of amine dehydrogenase through a Sephadex G-1 (2.6 by 4 cm) column. The crude extract was obtained from NPA-grown cells and contained 21 mg ofprotein. The amine dehydrogenase was eluted in 67 mm phosphate buffer (ph 7.5) and collected in 1-mI fractions. The void volume of the column was determined with blue dextran, and amine dehydrogenase (A) and absorbance at 28 nm () were measured. a.5 F 41.3 F.21F.1 I I I I I I MOLECULAR WEIGHT x 1 4 FIG. 3. Chromatography of amine dehydrogenase from NPA-grown cells of M. convolutum strain NPA-1 on a Sephadex G-1 (2.6 by 4 cm). The amine dehydrogenase was partially purified by previous gel filtration and applied to the column that was precalibrated with marker proteins. The markers used and molecular weights were: (1) ribonuclease A, 13,7; (2) chymotrypsinogen A, 25,; (3) ovalbumin, 45,; and (4) aldolase, 158,. (A) The point at which amine dehydrogenase was eluted from the column. The void volume was determined with blue dextran. K.,g for each protein standard was determined by Ve - Vo/Vt - Vo, where Ve is elution volume for the protein, Vo is void volume, and Vt is total bed volume. I 1- z

5 VOL. 124, 1975 in the utilization of NPA. The significant incorporation of "'C2 into the pyruvate produced by nonproliferating cells of strain NPA-1 during the oxidation of NPA and propionate indicated that these compounds were utilized via the methyl-malonate to succinate pathway (2). The growth of M. convolutum on propionate or NPA also yielded cells with a predominance (94.7%) of odd chain fatty acids (Table 3). The incorporation of propionate as a primer in fatty acid synthesis would account for this predominance of odd chain fatty acids. Previous studies (1, 11, 18) suggest that the fatty acid composition of hydrocarbon-grown bacteria often reflects the metabolic pathways involved in utilization of the substrate. The marked increase in odd chain fatty acids in cells after growth on propionate has also been shown in Bacillus subtilis (9) and Mycobacterium vaccae (18). The presence of isocitrate lyase in M. convolutum strain NPA-1 after growth on IPA and PD and malonate (Table 1) and a predominance of even-carbon fatty acids in the organism after growth on these substrates (Table 3) suggest that IPA, PD, and malonate are metabolized through a 2-carbon intermediate. Results in Table 4 indicate that the enzyme(s) involved in deamination of NPA, IPA, and PD was inducible since it was not present in cells grown on glucose or propionate. Amine dehydrogenase activity was located in the supernatant fraction after high-speed centrifugation and it is apparent that the amine dehydrogenase in M. convolutum strain NPA-1 is a soluble enzyme. This enzyme cannot oxidize NPA, IPA, and PD in the absence of an electron acceptor such as phenazine methyl sulfate. The enzyme will not react with oxygen and it is assumed that the electron acceptor for this enzyme would be flavin or pyridoxal derivatives as suggested for the amine dehydrogenase in Pseudomonas AM-1 (7, 8). This is in contrast to the amine oxidase present in Pseudomonas sp MS (12) and Pseudomonas aminovorans (3). M. convolutum strain NPA-1 after growth on WIA readily utilized both WPA and NPA, but NPA-grown cells did not oxidize IPA without a measurable lag. This lag was not evident with crude extracts suggesting that the induction of a transport system for IPA is necessary. A single amine dehydrogenase activity peak was detected after Sephadex G-1 chromatography of crude cell extracts of NPA-grown cells (Fig. 2) and similar results were obtained with IPA-grown cells. When these fractions were pooled and analyzed by polyacrylamide gel AMINE METABOLISM IN M. CONVOLUTUM 289 electrophoresis there was one major band of enzyme activity (Fig. 3) indicating that one enzyme of broad specificity is involved in the oxidation of NPA, IPA, and PD. LITERATURE CITED 1. Beam, H. W., and J. J. Perry Microbial degradation of cycloparaffinic hydrocarbons via co-metabolism and commensalism. J. Gen. Microbiol. 82: Blevins, W. T., and J. J. Perry Metabolism of propane, n-propylamine, and propionate by hydrocarbon-utilizing bacteria. J. Bacteriol. 112: Boulton, C. A., M. J. C. Crabble, and P. J. Large Microbial oxidation of amines. Partial purification of a trimethylamine mono-oxygenase from Pseudomonas aminovorans and its role in growth on trimethylamine. Biochem. J. 14: Carroll, K. K Quantitative estimation of peak areas in gas-liquid chromatograph. Nature (London) 191: Davis, B. J Disc electrophoresis. II. Method and application to human serum proteins. Ann. N. Y. Acad. Sci. 121: Dunlap, K. R., and J. J. Perry Effect of substrate on the fatty acid composition of hydrocarbon-utilizing microorganisms. J. Bacteriol. 94: Eady, R. R., and P. J. Large Purification and properties of an amine dehydrogenase from Pseudomonas AM-1 and its role in growth on methylamine. Biochem. J. 16: Eady, R. R., and P. J. Large Microbial oxidation of amines. 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