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Incorporation of 18O into long-chain, secondary alcohols derived from ester mycolic acids in Mycobacterium phlei
421
Biochimica et Biophysics Acta, 712 (1982) 427-429 Elsevier Biomedical Press
BBA Report
BBA 50013
INCORPORATION ESTER MYCOLIC SEIKO TORIYAMA
OF I80 INTO LONG-CHAIN, SECONDARY ACIDS IN MYCOBACTZWUM PHLEI
a, SADAO IMAIZUMI a, IKUKO TOMIYASU
a Department of Bacteriology, Niigata University School of Medicine, Osaka City University Medical School, Asahimachi-I, Osaka (Japan)
ALCOHOLS
DERIVED FROM
b, MASAMIKI MASUI b and IKUYA YANO W* Asahimachi-dori-I,
Niigata
and b Department
of Bacteriology,
(Received March 5th, 1982)
Key words: Estermycolic
acirl; Baeyer- Villiger oxidation;
Oxygen incorporation; (Mycobacterium
phlei)
I80 from an 1802 atmosphere was actively incorporated into long-chain, secondary alcohols, such as 2-octadecanol, 2-eicosanol and 2-docosanol, which are derived from ester mycolic acids, by short-term incubation of the growing cell suspension of Mycobacterium p/de;. Under the same conditions, the absence of significant ions due to 180 incorporation into dicarboxy mycolic acids, which are also derived from ester mycolic acids, indicated that the ester mycolic acids were synthesized from keto mycolic acids by a reaction which is biologically equivalent to Baeyer-Villiger-type oxidation.
Mycolic acids are the most characteristic components of cell wall lipids in Mycobacteria and related taxa; they contribute to the physiological properties of cell walls, such as acid-fastness or hydrophobicity. Analytical studies on mycolic acids have been carried out intensively in past decades and reveal that there are many subclasses or molecular species differing in polarity, double bond numbers and carbon chain lengths. cy-Mycolic acids, the most widely distributed species among Mycobacteria, Nocardia and Corynebacteria, are well known to be cu-alkyl branched-chain P-hydroxy fatty acids [l]. On the other hand, the occurrence of dicarboxy mycolic acids, a more polar subclass of mycolic acids, has been reported to occur among some species of Mycobacteria [2-91 and these acids were postulated to be derived from ester mycolic acids which could be synthesized by enzymatic oxidation of fi-mycolic acid (monoketo monohydroxy mycolic acids) [6].
l
To whom correspondence should be addressed.
OOOS-2760/82/OOOC-0000/$02.75
0 1982 Elsevier Biomedical Press
However, evidence for ester or dicarboxy mycolic acid biosynthesis has not yet been demonstrated. Recently, we have shown that M. phlei, a chromogenic rapidly growing species, possessed cymycolic, /3-(keto) mycolic and dicarboxy mycolic acids as major components of both the extractable and cell wall-bound lipids; the total amount and molecular species composition were affected by the growth temperature [15,16]. Since the cells of M. phlei grown at higher temperatures produced dicarboxy mycolic acid and secondary alcohols abundantly, both of which are components of ester mycolic acids in situ, we examined the biosynthetic route of the above-mentioned mycolic acids using “0,. Gas chromatographic-mass spectrometric analysis of secondary alcohols and dicarboxy mycolic acids after incubation of the cells in an 180, atmosphere revealed that the I80 was incorporated into the secondary alcohols, but not into dicarboxy mycolic acids. From these results, the biosynthesis of dicarboxy mycolic acids or ester mycolic acids may be considered analogous
428
to the Baeyer-Villiger oxidations of keto mycolic acids by peracids. M. phlei was grown in a medium containing 1.0% glucose, 0.5% peptone and 0.2% yeast extract at pH 7.0. At the mid-log stage of growth (48 h at 4O”C), cells were harvested and resuspended in the fresh medium in loo-ml Erlenmeyer flasks with glass-sealed stoppers and vacuum cocks. To each vessel 50 ml cell suspension containing 0.05 g of packed cells were added and N, gas was flushed through an ice bath to remove the 1602. “0, (99.0%, Bokusui Brown Co., Ltd.) and N, were introduced from the cock and adjusted to give a concentration of approximately 20.0% 0,. Reactions were started by the incubation of sealed cell suspension at 45°C. After shaking vigorously for 4- 14 h, the reactions were stopped by the addition of an equal volume of 10.0% KOH/methanol and the mixture was hydrolyzed for 3 h under reflux. The reaction mixtures were extracted with n-hexane to obtain long-chain compounds, and then the fatty acids were transmethylated with benzene/ methanol/sulfuric acid (10 : 20 : 1, v/v). The resultant fatty acid methyl esters were extracted and separated by TLC on silica gel G with a solvent of n-hexane/diethyl ether (4 : 1, v/v). After the plates were visualized with iodine vapor, dicarboxy mycolic acid methyl esters and secondary alcohols were recovered from the thin-layer plates and tri-
Octadecunol-2 M-15 h/z3271
Eicosanol- 2 M-15 (ma3551
Dowsanol- 2 M-15 Im/rJBJJ
IO
02
enriched lj4454*2
L
Fig. 1. Partial mass spectra of secondary alcohols obtained from the cells incubated in 1602- or ‘*O,-enriched atmosphere. (M - 15) and neighboring ions due to I80 incorporation are reproduced partially as bar graphs.
methylsilylated with pyridine/bistrimethylsilyl trifluoroacetoamide (2: 1, v/v) for 20 min at 70°C. The resultant trimethylsilyl derivatives of dicarboxy mycolic acid methyl esters and secondary alcohols were subjected to gas chromatographymass spectrometry (Hitachi M-60) with a glass column of Diasolid ZT at 320 or 16O”C, respectively, as reported previously [ 161. Mass spectra were recorded at 20 eV of ionizing energy with the accelerating voltage of 3.2 kvolts. Fig. I shows a partial mass spectrum of trimethylsilyl derivatives of secondary alcohols obtained after incubation of M. phlei in a 1602- or “0,-enriched atmosphere for 14 h. As reported previously, the most prominent ion of trimethylsilyated secondary alcohol was (M - 15) ions instead of molecular ions (M)+ [ 161. On the other hand, the position of hydroxyl group of secondary alcohols was indicated by the fragment ions due to cleavage between the C-C bond containing the oxytrimethylsilyl group (m/z 117 and m/z( M - 15)) [16]. Since gas chromatographic analysis showed the occurrence of three major compounds, octadecanol-2, eicosanol-2 and docosanol-2, (M 15) ions due to loss of a methyl group from molecular ions of three compounds were analyzed in detail. Both the mass spectra of trimethylsilylated secondary alcohols from the cells grown in 1602 and “0, atmospheres showed a marked difference in the fragment ions containing the hydroxyl group. (M - 15) + 2 ions of trimethylsilylated octadecanol-2 (m/z 329) were 7.7% of (M - 15) ions in the cells incubated in 1602 atmosphere, while they were 88.2% in cells incubated in ‘*O,-enriched atmosphere. Similar results were obtained for eicosanol-2 and docosanol-2, with less enrichment. In order to determine quantitatively the incorporation rates of 180 into the secondary alcohols, mass fragmentographic analysis was performed with monitering ions of (M - 15), (M 15)-t 1, (M15)+2 and (MIS)+ 3, and the amount of enriched ions was calculated from peak areas, as indicated in TableI. It was found that I80 atoms were incorporated into all the secondary alcohols of this bacterium, with the most abundant enrichment for octadecanol-2. On the other hand, trimethylsilylated dicarboxy mycolic acid methyl esters were separated by GC/MS and their (M15) ions were analyzed in detail. How-
429 TABLE I SHIFTS AND ENRICHMENT OF (M- 15) IONS OF TRIMETHYLSILYLATED OBTAINED AFTER INCUBATION IN l8O2 ATMOSPHERE
SECONDARY ALCOHOLS FROM M.
PHLEI
Values are expressed as percentages of intensities for (M - 15) ions. Docosanol-2
Eicosanol-2
Octadecanol-2 ‘80
‘60
‘80
160
‘80
I60
(M-15)+1 (M-15)
34.3
28.4
32.1
27.6
34.0
31.3
(M-15)+2 (M-15)
88.2
7.7
32.1
9.2
30.7
6.9
(M-15)+3 (M-15)
22.1
4.1
10.1
3.3
12.3
-
ever, no significant enrichment of (M - 15) + 2 and (M - 15) + 3 ions due to I80 incorporation was observed. From the results obtained above, it was demonstrated that molecular oxygen was the source of oxygen used for the oxygenation reaction in the conversion of ketomycolic acid to ester mycolic acid. A similar oxidation reaction of 2butanone or 2-tridecanone by Nocardia sp. or Pseudomonas cepacia, has been reported to occur by way of ester intermediates, which are hydrolyzed to alcohol and acid [lo- 141. We are now trying to prepare cell-free extracts which catalyze the oxygenase reaction of ketomycolic acids. References 1 Asselineau, J. (1966) The Bacterial Lipids, Holden-Day Inc., San Francisco 2 Clermont, R. and Lederer, E. (1956) Compt. Rend. Acad. Sci. 242, 2600-2603 3 Markovits, J., Pinte, R. and Etemadi, A.H. (1966) C.R. Acad. Sci. (C), 263, 960-962
4 Miquel, A.M., Ginsburg, H. and Asselineau, J. (1963) Bull. Sot. Chim. Biol., 45, 715-730 5 Lanklle, M.G. (1963) Compt. Rend. Acad. Sci. 257, 781783 6 Etemadi, A.H. and Gasche, J. (1965) Bull. Sot. Chim. Biol., 47, 2095-2104 7 Lane&e, M.A. (1969) These Doctorat, Toulouse 8 Laneelle, M.A. and Laneelle, G. (1970) Eur. J. B&hem. 12, 296-300 9 Prome, J.C., Lacave, C., Ahibo-Coffy, A. and Savagnac, A. (1976) Eur. J. B&hem. 63, 543-552 10 Fomey, F.W. and Markovetz, A.J. (1968) J. Bacterial. 96, 1055-1064 11 Fomey, F.W. and Markovetz, A.J. (1969) B&hem. Biophys. Res. Commun. 37, 31-38 12 Rahim, M.A. and Sih, C.J. (1966) J. Biol. Chem. 241, 3615-3623 13 B&ton, L.N. and Markovetz, A.J. (1974) B&him. Biophys. Acta. 369, 45-49 14 B&ton, L.N. and Markovets, A.J. (1977) J. Biol. Chem. 252, 8561-8566 15 Toriyama, S., Yano, I., Masui, M., Kusunose, M. and Kusunose, E. (1978) FEBS Lett. 95, 11l-l 15 16 Toriyama, S., Yano, I., Masui, M., Kusunose, E. and Kusunose, M. (1980) J. B&hem. 88,211-221
Biochimica et Biophysics Acta, 712 (1982) 427-429 Elsevier Biomedical Press
BBA Report
BBA 50013
INCORPORATION ESTER MYCOLIC SEIKO TORIYAMA
OF I80 INTO LONG-CHAIN, SECONDARY ACIDS IN MYCOBACTZWUM PHLEI
a, SADAO IMAIZUMI a, IKUKO TOMIYASU
a Department of Bacteriology, Niigata University School of Medicine, Osaka City University Medical School, Asahimachi-I, Osaka (Japan)
ALCOHOLS
DERIVED FROM
b, MASAMIKI MASUI b and IKUYA YANO W* Asahimachi-dori-I,
Niigata
and b Department
of Bacteriology,
(Received March 5th, 1982)
Key words: Estermycolic
acirl; Baeyer- Villiger oxidation;
Oxygen incorporation; (Mycobacterium
phlei)
I80 from an 1802 atmosphere was actively incorporated into long-chain, secondary alcohols, such as 2-octadecanol, 2-eicosanol and 2-docosanol, which are derived from ester mycolic acids, by short-term incubation of the growing cell suspension of Mycobacterium p/de;. Under the same conditions, the absence of significant ions due to 180 incorporation into dicarboxy mycolic acids, which are also derived from ester mycolic acids, indicated that the ester mycolic acids were synthesized from keto mycolic acids by a reaction which is biologically equivalent to Baeyer-Villiger-type oxidation.
Mycolic acids are the most characteristic components of cell wall lipids in Mycobacteria and related taxa; they contribute to the physiological properties of cell walls, such as acid-fastness or hydrophobicity. Analytical studies on mycolic acids have been carried out intensively in past decades and reveal that there are many subclasses or molecular species differing in polarity, double bond numbers and carbon chain lengths. cy-Mycolic acids, the most widely distributed species among Mycobacteria, Nocardia and Corynebacteria, are well known to be cu-alkyl branched-chain P-hydroxy fatty acids [l]. On the other hand, the occurrence of dicarboxy mycolic acids, a more polar subclass of mycolic acids, has been reported to occur among some species of Mycobacteria [2-91 and these acids were postulated to be derived from ester mycolic acids which could be synthesized by enzymatic oxidation of fi-mycolic acid (monoketo monohydroxy mycolic acids) [6].
l
To whom correspondence should be addressed.
OOOS-2760/82/OOOC-0000/$02.75
0 1982 Elsevier Biomedical Press
However, evidence for ester or dicarboxy mycolic acid biosynthesis has not yet been demonstrated. Recently, we have shown that M. phlei, a chromogenic rapidly growing species, possessed cymycolic, /3-(keto) mycolic and dicarboxy mycolic acids as major components of both the extractable and cell wall-bound lipids; the total amount and molecular species composition were affected by the growth temperature [15,16]. Since the cells of M. phlei grown at higher temperatures produced dicarboxy mycolic acid and secondary alcohols abundantly, both of which are components of ester mycolic acids in situ, we examined the biosynthetic route of the above-mentioned mycolic acids using “0,. Gas chromatographic-mass spectrometric analysis of secondary alcohols and dicarboxy mycolic acids after incubation of the cells in an 180, atmosphere revealed that the I80 was incorporated into the secondary alcohols, but not into dicarboxy mycolic acids. From these results, the biosynthesis of dicarboxy mycolic acids or ester mycolic acids may be considered analogous
428
to the Baeyer-Villiger oxidations of keto mycolic acids by peracids. M. phlei was grown in a medium containing 1.0% glucose, 0.5% peptone and 0.2% yeast extract at pH 7.0. At the mid-log stage of growth (48 h at 4O”C), cells were harvested and resuspended in the fresh medium in loo-ml Erlenmeyer flasks with glass-sealed stoppers and vacuum cocks. To each vessel 50 ml cell suspension containing 0.05 g of packed cells were added and N, gas was flushed through an ice bath to remove the 1602. “0, (99.0%, Bokusui Brown Co., Ltd.) and N, were introduced from the cock and adjusted to give a concentration of approximately 20.0% 0,. Reactions were started by the incubation of sealed cell suspension at 45°C. After shaking vigorously for 4- 14 h, the reactions were stopped by the addition of an equal volume of 10.0% KOH/methanol and the mixture was hydrolyzed for 3 h under reflux. The reaction mixtures were extracted with n-hexane to obtain long-chain compounds, and then the fatty acids were transmethylated with benzene/ methanol/sulfuric acid (10 : 20 : 1, v/v). The resultant fatty acid methyl esters were extracted and separated by TLC on silica gel G with a solvent of n-hexane/diethyl ether (4 : 1, v/v). After the plates were visualized with iodine vapor, dicarboxy mycolic acid methyl esters and secondary alcohols were recovered from the thin-layer plates and tri-
Octadecunol-2 M-15 h/z3271
Eicosanol- 2 M-15 (ma3551
Dowsanol- 2 M-15 Im/rJBJJ
IO
02
enriched lj4454*2
L
Fig. 1. Partial mass spectra of secondary alcohols obtained from the cells incubated in 1602- or ‘*O,-enriched atmosphere. (M - 15) and neighboring ions due to I80 incorporation are reproduced partially as bar graphs.
methylsilylated with pyridine/bistrimethylsilyl trifluoroacetoamide (2: 1, v/v) for 20 min at 70°C. The resultant trimethylsilyl derivatives of dicarboxy mycolic acid methyl esters and secondary alcohols were subjected to gas chromatographymass spectrometry (Hitachi M-60) with a glass column of Diasolid ZT at 320 or 16O”C, respectively, as reported previously [ 161. Mass spectra were recorded at 20 eV of ionizing energy with the accelerating voltage of 3.2 kvolts. Fig. I shows a partial mass spectrum of trimethylsilyl derivatives of secondary alcohols obtained after incubation of M. phlei in a 1602- or “0,-enriched atmosphere for 14 h. As reported previously, the most prominent ion of trimethylsilyated secondary alcohol was (M - 15) ions instead of molecular ions (M)+ [ 161. On the other hand, the position of hydroxyl group of secondary alcohols was indicated by the fragment ions due to cleavage between the C-C bond containing the oxytrimethylsilyl group (m/z 117 and m/z( M - 15)) [16]. Since gas chromatographic analysis showed the occurrence of three major compounds, octadecanol-2, eicosanol-2 and docosanol-2, (M 15) ions due to loss of a methyl group from molecular ions of three compounds were analyzed in detail. Both the mass spectra of trimethylsilylated secondary alcohols from the cells grown in 1602 and “0, atmospheres showed a marked difference in the fragment ions containing the hydroxyl group. (M - 15) + 2 ions of trimethylsilylated octadecanol-2 (m/z 329) were 7.7% of (M - 15) ions in the cells incubated in 1602 atmosphere, while they were 88.2% in cells incubated in ‘*O,-enriched atmosphere. Similar results were obtained for eicosanol-2 and docosanol-2, with less enrichment. In order to determine quantitatively the incorporation rates of 180 into the secondary alcohols, mass fragmentographic analysis was performed with monitering ions of (M - 15), (M 15)-t 1, (M15)+2 and (MIS)+ 3, and the amount of enriched ions was calculated from peak areas, as indicated in TableI. It was found that I80 atoms were incorporated into all the secondary alcohols of this bacterium, with the most abundant enrichment for octadecanol-2. On the other hand, trimethylsilylated dicarboxy mycolic acid methyl esters were separated by GC/MS and their (M15) ions were analyzed in detail. How-
429 TABLE I SHIFTS AND ENRICHMENT OF (M- 15) IONS OF TRIMETHYLSILYLATED OBTAINED AFTER INCUBATION IN l8O2 ATMOSPHERE
SECONDARY ALCOHOLS FROM M.
PHLEI
Values are expressed as percentages of intensities for (M - 15) ions. Docosanol-2
Eicosanol-2
Octadecanol-2 ‘80
‘60
‘80
160
‘80
I60
(M-15)+1 (M-15)
34.3
28.4
32.1
27.6
34.0
31.3
(M-15)+2 (M-15)
88.2
7.7
32.1
9.2
30.7
6.9
(M-15)+3 (M-15)
22.1
4.1
10.1
3.3
12.3
-
ever, no significant enrichment of (M - 15) + 2 and (M - 15) + 3 ions due to I80 incorporation was observed. From the results obtained above, it was demonstrated that molecular oxygen was the source of oxygen used for the oxygenation reaction in the conversion of ketomycolic acid to ester mycolic acid. A similar oxidation reaction of 2butanone or 2-tridecanone by Nocardia sp. or Pseudomonas cepacia, has been reported to occur by way of ester intermediates, which are hydrolyzed to alcohol and acid [lo- 141. We are now trying to prepare cell-free extracts which catalyze the oxygenase reaction of ketomycolic acids. References 1 Asselineau, J. (1966) The Bacterial Lipids, Holden-Day Inc., San Francisco 2 Clermont, R. and Lederer, E. (1956) Compt. Rend. Acad. Sci. 242, 2600-2603 3 Markovits, J., Pinte, R. and Etemadi, A.H. (1966) C.R. Acad. Sci. (C), 263, 960-962
4 Miquel, A.M., Ginsburg, H. and Asselineau, J. (1963) Bull. Sot. Chim. Biol., 45, 715-730 5 Lanklle, M.G. (1963) Compt. Rend. Acad. Sci. 257, 781783 6 Etemadi, A.H. and Gasche, J. (1965) Bull. Sot. Chim. Biol., 47, 2095-2104 7 Lane&e, M.A. (1969) These Doctorat, Toulouse 8 Laneelle, M.A. and Laneelle, G. (1970) Eur. J. B&hem. 12, 296-300 9 Prome, J.C., Lacave, C., Ahibo-Coffy, A. and Savagnac, A. (1976) Eur. J. B&hem. 63, 543-552 10 Fomey, F.W. and Markovetz, A.J. (1968) J. Bacterial. 96, 1055-1064 11 Fomey, F.W. and Markovetz, A.J. (1969) B&hem. Biophys. Res. Commun. 37, 31-38 12 Rahim, M.A. and Sih, C.J. (1966) J. Biol. Chem. 241, 3615-3623 13 B&ton, L.N. and Markovetz, A.J. (1974) B&him. Biophys. Acta. 369, 45-49 14 B&ton, L.N. and Markovets, A.J. (1977) J. Biol. Chem. 252, 8561-8566 15 Toriyama, S., Yano, I., Masui, M., Kusunose, M. and Kusunose, E. (1978) FEBS Lett. 95, 11l-l 15 16 Toriyama, S., Yano, I., Masui, M., Kusunose, E. and Kusunose, M. (1980) J. B&hem. 88,211-221