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Quinones of brevibacterium
Biochimica et Biophysics Acta, 348 (1974) 162-165 0 Elsevier Scientific Publishing Company, Amsterdam
- Printed in The Netherlands
BBA 56419
QUINONES OF BREVIBACTERIUM
T. KANZAKI,
Y. SUGIYAMA*,
K. KITANO, Y. ASHIDA*
and I. IMADA**
Microbiological Research Laboratories, Central Research Division, Takeda Chemical Industries Ltd, Osaka 532 (Japan) (Received November
rzth, 1973)
SUMMARY
Two unusual menaquinones have been revealed during examination of Brevibacterium thiogenitalis and Brevibacterium vitarumen. Both microorganisms were found to contain menaquinones-8 (II-H2) and -9 (II-HZ). The major quinone of the former was menaquinone-9 (II-H2) while that of the latter was menaquinone-8 (II-H& No ubiquinones were demonstrated in both microorganisms.
Previous papers [I-31 demonstrated that the biosynthesis of L-glutamate in Brevibacterium and other microorganisms was regulated by the copper ion concentrations in the medium when those organisms were cultivated with acetate as the sole source of carbon, and this was due to changes in their electron transport systems and those in the coupled oxidative phosphorylation efficiency which depends upon the copper ion concentrations in the medium. Thus an attempt was made to determine the kinds of quinones which are known to play a role in the electron transport systems [4, 51 and have not yet been demonstrated in any of the microorganisms belonging to Brevibacterium. Brevibacterium thiogenitalis D-248, an L-glutamate-producing unsaturated fatty-acid auxotroph [6], was grown in the acetate medium [2] for 24 h. Wet cells (134 g) were extracted with three 5oo-ml portions of ethanol at 60 “C for I h. The extracts were combined and extracted three times with n-hexane (750 ml) after the addition of water ( IOO ml). The combined n-hexane layer was then worked up in the usual manner to give 0.97 g of yellow oil. This oil was dissolved in chloroform (3 ml) and adsorbed on a silicic acid column (20 g, 2.5 x 14 cm). Elution with chloroform (50 ml) yielded 0.126 g of yellow oil. A solution of the yellow oil in benzene (0.5 ml) was chromatographed on thin-layer plates (20 cm x 20 cm, IO g of silica gel GF,,,, E. Merck) in benzene-ethylacetate (9:1, v/v). The resulting yellow band at RF 0.8 was eluted with ether and worked up in the usual manner to obtain the orange-coloured Abbreviations: Menaquinone-n (II-H& a menaquinone with a reduced double bond in the second unit of n prenyl units, counting out from the nucleus. * Present address: Technological Department, Takasago Plant, Takeda Chemical Industries Ltd, Takasago, Hyogo, Japan. ** Present address: Chemical Research Laboratories, Central Research Division, Takeda Chemical Industries Ltd, Osaka, Japan.
I63
oil as a quinone fraction. The solution of the q&one fraction in acetone (0.3 ml) was applied to a reversed-phase thin-layer chromatography by the use of thin-layer plates impregnated with paraffin and developed in acetone-water (97: 3, v/v), because the homologues which are different in the isoprenoid side chain are hardly separated by the thin-layer chromatography described above [7]. Yellow quinone bands at RF 0.42 and 0.55 were scraped ofI. Each silica-gel portion was washed with n-hexane and then extracted with ether. The ether extract yielded 20 mg and 4 mg of orange-coloured oils from RF 0.42 and RF 0.55 fractions, respectively. They similarly showed ultraviolet absorption maxima at 245, 250, 264, 272 and 332 nm and infrared absorption structure. peaks at 16oo,1620 and 1660 cm-‘, suggesting a 2,3-~alkylnaphthoq~none The structure of the RF 0.42 compound was confirmed to be menaquinone-9 (II-H,) by the superimposability of its NMR spectrum (Table I) and mass spectrum (Fig. I) with those described previously [8,9]. The RF 0.55 compound was similarly identified as menaquinone-8 (II-H,) on the basis of NMR and mass spectra as shown in Table I and Fig, 2. In addition to these quinones, two faint yellow bands were observed on the above reversed-phase chromatography. There may be trace amounts of two more menaquinones in the cells, since the extracts of the bands showed similar ultraviolet absorption maxima to those of the above quinones. TABLE I NMR SPECTRA OF MENAQUINONES The bands refer to IOO MHz in carbon tetrachloride, (Varian HA-100).
using tetramethylsilane
as an internal standard
r-value
CH3
CHz,CH CH3C=(rnzns) CH,C=(cis) CH,C=(3’-mm) CH&= Quinone-CH3
QuinoneCH2 CH= Aromatic-H
RF 0.55 Compound [Menaquinone-8 (H-H,)]
RI; 0.42 Compound [Menaquinone-9 (II-Hz)]
9.17 8.6-8.85 8.42 8.35 8.24 8.03 7.85 6.70 4.98 2.0-2.43
9.17 8.65-8.85 8.42 8.35 8.23 8.03 7.85 6.70 4.97 r.99-2.41
(d*, (b, (s, (s, (s, (b, ts, (d, (bb, (m.
3H*) 7H) r8H) 3H) 3H) 24H) 3H) 2H) 7H) 4H)
(d, (b, (s, (a, :,
3H) 7H)
21H) 3H) 2::;
;, 2:; (b, 8H) (m, 4H)
* s, singlet; d, doublet; m, multiplet; b, broad. ** Proton number.
When 45.2 g (wet wt) of B. thi~ge~ita~isD-248 ce& grown in the glucose medium [I] were treated in the same way as above, 2 mg of menaquinone-8 (II-H,) and IO mg of menaquinone-9 (II-H,) were obtained. From this fact, main menaquinones were found to remain qualitatively and quantitatively invariable even if the carbon source for cultivation varied, that is, despite changes in the ener~-yielding systems. Incidentally, cells of Brevibacterium vitarumen IF0 12143, an organism not producing L-glutamate ,were examined on menaquinones. Cultivation of B. vitarumen IF0 I 2143 was carried out in the glucose medium [I] from which oleate was excluded. The wet cells (557 g) were treated similarly to the case of B. thioge~itazi~. As a result,
0
I
513
~272,273t204,d-d3&,137
581
t
Fig. 2. Mass spectrum of menaquinone-8 (II I&). The spectrum was recorded on JMS-OISC (Japan Electron Optics Lab.) at an ionization potential of 75 eV, a sample temperature of 210 “C and a chamber temperature of 230 ‘C. Since m/e 408 was not observed, the last diallylic bond occurred between the prenyl units 3 and 4, and the saturated double bond was found to be located in the second prenyl unit, counting out from the quinone ring [g].
44.5
&OS41
Fig. I. Mass spectrum of menaquinoneg (II-I&). The spectrum was recorded on an Hitachi RMU- 6D at an ionization potential of 75 eV, with sample and chamber temperatures of 200 “C. Fragment peaks were those referred to in refs. 8 and 9.
;i; P
165
280 mg of menaquinone-8 (II-H,) and 70 mg of menaquinone-g (II-H,) were obtained. The benzene solutions of the yellow oils obtained from the n-hexane extracts in three cases described above were chromatographed with benzene on the thin-layer plates and the portions corresponding to ubiquinone homologues (ubiquinone-6 to -IO) were extracted with ether. Ubiquinone homologues were not detected in those extracts by ultraviolet spectra and spraying with the leucomethylene blue reagent [IO]. These results suggest the universal presence of menaquinone-8 (II-H,) and -9 (II-H,) in microorganisms of Brevibacterium. Though this is the first demonstration of the presence of menaquinones in Brevibacterium, it has been reported that Corynebacterium, which is closely allied to Brevibacterium, contains menaquinones and no ubiquinones. Thus this finding was consistent with the general knowledge [II] that Gram-positive bacteria contain menaquinones. Menaquinones with partially saturated side chains have been found in microorganisms belonging to Mycobacterium 1121, Streptomyces [13], Micrococcaceue [14] and Corynebacterium [IS], and those of Mycobacterium phlei proved to take part in oxidative phosphorylation [4]. Menaquinones of Brevibacterium might play the same role as in the electron transport of M. phlei. ACKNOWLEDGEMENTS We thank Drs S. Tatsuoka, R. Takeda, H. Morimoto, M. Isono, H. Fukuda and Mr K. Takeba for their kind encouragement and reliable advices. We are also indebted to Messrs. T. Shima and A. Ouchida, and Miss F. Kasahara for the measurement of physicochemical data. REFERENCES I Kanzaki, T., Nakatsui, I., Kitano, K., Sugiyama, Y., Nishio, M. and Ishikawa, M. (1973) Agric. Biol. Chem. 37, 1407-1416 2 Sugiyama, Y., Kitano, K. and Kanzaki, T. (1973) Agric. Biol. Chem. 37, 1607-1612 3 Sugiyama, Y., Kitano, K. and Kanzaki, T. (1973) Agric. Biol. Chem. 37, 1837-1847 4 Dunphy, P. J., Gutnick, D. L., Phillips, P. G. and Brodie, A. F. (1968) J. Biol. Chem. 243, 398407 Fujita, M., Ishikawa, S. and Shimazono, N. (1966) J. Biochem. Tokyo 59, 104-114 2 Kanzaki, T., Isobe, K., Okazaki, H., Motizuki, K. and Fukuda, H. (1967) Agric. Biol. Chem. 31, 13o7-r313 7 Dunphy, P. J. and Brodie, A. F. (1971) in Methods in Enzymology (McCormick, D. B. and Wright, L. D., eds), Vol. XVIIIC, pp. 407-461, Academic Press, New York 8 Beau, S., Azerad, R. and Lederer, E. (1966) Bull Sot. Chim. Biol. 48, 569-581 9 Campbell, I. M. and Bentley, R. (1968) Biochemistry 7, 3323-3327 IO Linn, B. O., Page, Jr, A. C., Wong, E. L., Gale, P. H., Shunk, C. H. and Folkers, K. (1959) J. Am. Chem. Sot. 81, 4007-4010 II Bishop, D. H. L., Pandya, K. P. and King, H. K. (1962) Biochem. J. 83, 606-614 _ __ _ - . - __ . . 12 GaIe, P. H., Arison, B. H., TreMer, N. R., Page, Jr, A. C. and Folkers, K. (1963) Biochemistry 2, 2oo-203 I3 Jeffries, L., Cawthome, M. A., Harris, M., Diplock, A. T., Green, J. and Price, S. A. (1967) Nature 215, 257-259 14 Phillips, P. G., Dunphy, P. J., Servis, K. L. and Brodie, A. F. (1969) Biochemistry 8,2856-2861 I5 Scholes, P. B. and King, H. K. (1965) Biochem. 5. 97, 766-768
- Printed in The Netherlands
BBA 56419
QUINONES OF BREVIBACTERIUM
T. KANZAKI,
Y. SUGIYAMA*,
K. KITANO, Y. ASHIDA*
and I. IMADA**
Microbiological Research Laboratories, Central Research Division, Takeda Chemical Industries Ltd, Osaka 532 (Japan) (Received November
rzth, 1973)
SUMMARY
Two unusual menaquinones have been revealed during examination of Brevibacterium thiogenitalis and Brevibacterium vitarumen. Both microorganisms were found to contain menaquinones-8 (II-H2) and -9 (II-HZ). The major quinone of the former was menaquinone-9 (II-H2) while that of the latter was menaquinone-8 (II-H& No ubiquinones were demonstrated in both microorganisms.
Previous papers [I-31 demonstrated that the biosynthesis of L-glutamate in Brevibacterium and other microorganisms was regulated by the copper ion concentrations in the medium when those organisms were cultivated with acetate as the sole source of carbon, and this was due to changes in their electron transport systems and those in the coupled oxidative phosphorylation efficiency which depends upon the copper ion concentrations in the medium. Thus an attempt was made to determine the kinds of quinones which are known to play a role in the electron transport systems [4, 51 and have not yet been demonstrated in any of the microorganisms belonging to Brevibacterium. Brevibacterium thiogenitalis D-248, an L-glutamate-producing unsaturated fatty-acid auxotroph [6], was grown in the acetate medium [2] for 24 h. Wet cells (134 g) were extracted with three 5oo-ml portions of ethanol at 60 “C for I h. The extracts were combined and extracted three times with n-hexane (750 ml) after the addition of water ( IOO ml). The combined n-hexane layer was then worked up in the usual manner to give 0.97 g of yellow oil. This oil was dissolved in chloroform (3 ml) and adsorbed on a silicic acid column (20 g, 2.5 x 14 cm). Elution with chloroform (50 ml) yielded 0.126 g of yellow oil. A solution of the yellow oil in benzene (0.5 ml) was chromatographed on thin-layer plates (20 cm x 20 cm, IO g of silica gel GF,,,, E. Merck) in benzene-ethylacetate (9:1, v/v). The resulting yellow band at RF 0.8 was eluted with ether and worked up in the usual manner to obtain the orange-coloured Abbreviations: Menaquinone-n (II-H& a menaquinone with a reduced double bond in the second unit of n prenyl units, counting out from the nucleus. * Present address: Technological Department, Takasago Plant, Takeda Chemical Industries Ltd, Takasago, Hyogo, Japan. ** Present address: Chemical Research Laboratories, Central Research Division, Takeda Chemical Industries Ltd, Osaka, Japan.
I63
oil as a quinone fraction. The solution of the q&one fraction in acetone (0.3 ml) was applied to a reversed-phase thin-layer chromatography by the use of thin-layer plates impregnated with paraffin and developed in acetone-water (97: 3, v/v), because the homologues which are different in the isoprenoid side chain are hardly separated by the thin-layer chromatography described above [7]. Yellow quinone bands at RF 0.42 and 0.55 were scraped ofI. Each silica-gel portion was washed with n-hexane and then extracted with ether. The ether extract yielded 20 mg and 4 mg of orange-coloured oils from RF 0.42 and RF 0.55 fractions, respectively. They similarly showed ultraviolet absorption maxima at 245, 250, 264, 272 and 332 nm and infrared absorption structure. peaks at 16oo,1620 and 1660 cm-‘, suggesting a 2,3-~alkylnaphthoq~none The structure of the RF 0.42 compound was confirmed to be menaquinone-9 (II-H,) by the superimposability of its NMR spectrum (Table I) and mass spectrum (Fig. I) with those described previously [8,9]. The RF 0.55 compound was similarly identified as menaquinone-8 (II-H,) on the basis of NMR and mass spectra as shown in Table I and Fig, 2. In addition to these quinones, two faint yellow bands were observed on the above reversed-phase chromatography. There may be trace amounts of two more menaquinones in the cells, since the extracts of the bands showed similar ultraviolet absorption maxima to those of the above quinones. TABLE I NMR SPECTRA OF MENAQUINONES The bands refer to IOO MHz in carbon tetrachloride, (Varian HA-100).
using tetramethylsilane
as an internal standard
r-value
CH3
CHz,CH CH3C=(rnzns) CH,C=(cis) CH,C=(3’-mm) CH&= Quinone-CH3
QuinoneCH2 CH= Aromatic-H
RF 0.55 Compound [Menaquinone-8 (H-H,)]
RI; 0.42 Compound [Menaquinone-9 (II-Hz)]
9.17 8.6-8.85 8.42 8.35 8.24 8.03 7.85 6.70 4.98 2.0-2.43
9.17 8.65-8.85 8.42 8.35 8.23 8.03 7.85 6.70 4.97 r.99-2.41
(d*, (b, (s, (s, (s, (b, ts, (d, (bb, (m.
3H*) 7H) r8H) 3H) 3H) 24H) 3H) 2H) 7H) 4H)
(d, (b, (s, (a, :,
3H) 7H)
21H) 3H) 2::;
;, 2:; (b, 8H) (m, 4H)
* s, singlet; d, doublet; m, multiplet; b, broad. ** Proton number.
When 45.2 g (wet wt) of B. thi~ge~ita~isD-248 ce& grown in the glucose medium [I] were treated in the same way as above, 2 mg of menaquinone-8 (II-H,) and IO mg of menaquinone-9 (II-H,) were obtained. From this fact, main menaquinones were found to remain qualitatively and quantitatively invariable even if the carbon source for cultivation varied, that is, despite changes in the ener~-yielding systems. Incidentally, cells of Brevibacterium vitarumen IF0 12143, an organism not producing L-glutamate ,were examined on menaquinones. Cultivation of B. vitarumen IF0 I 2143 was carried out in the glucose medium [I] from which oleate was excluded. The wet cells (557 g) were treated similarly to the case of B. thioge~itazi~. As a result,
0
I
513
~272,273t204,d-d3&,137
581
t
Fig. 2. Mass spectrum of menaquinone-8 (II I&). The spectrum was recorded on JMS-OISC (Japan Electron Optics Lab.) at an ionization potential of 75 eV, a sample temperature of 210 “C and a chamber temperature of 230 ‘C. Since m/e 408 was not observed, the last diallylic bond occurred between the prenyl units 3 and 4, and the saturated double bond was found to be located in the second prenyl unit, counting out from the quinone ring [g].
44.5
&OS41
Fig. I. Mass spectrum of menaquinoneg (II-I&). The spectrum was recorded on an Hitachi RMU- 6D at an ionization potential of 75 eV, with sample and chamber temperatures of 200 “C. Fragment peaks were those referred to in refs. 8 and 9.
;i; P
165
280 mg of menaquinone-8 (II-H,) and 70 mg of menaquinone-g (II-H,) were obtained. The benzene solutions of the yellow oils obtained from the n-hexane extracts in three cases described above were chromatographed with benzene on the thin-layer plates and the portions corresponding to ubiquinone homologues (ubiquinone-6 to -IO) were extracted with ether. Ubiquinone homologues were not detected in those extracts by ultraviolet spectra and spraying with the leucomethylene blue reagent [IO]. These results suggest the universal presence of menaquinone-8 (II-H,) and -9 (II-H,) in microorganisms of Brevibacterium. Though this is the first demonstration of the presence of menaquinones in Brevibacterium, it has been reported that Corynebacterium, which is closely allied to Brevibacterium, contains menaquinones and no ubiquinones. Thus this finding was consistent with the general knowledge [II] that Gram-positive bacteria contain menaquinones. Menaquinones with partially saturated side chains have been found in microorganisms belonging to Mycobacterium 1121, Streptomyces [13], Micrococcaceue [14] and Corynebacterium [IS], and those of Mycobacterium phlei proved to take part in oxidative phosphorylation [4]. Menaquinones of Brevibacterium might play the same role as in the electron transport of M. phlei. ACKNOWLEDGEMENTS We thank Drs S. Tatsuoka, R. Takeda, H. Morimoto, M. Isono, H. Fukuda and Mr K. Takeba for their kind encouragement and reliable advices. We are also indebted to Messrs. T. Shima and A. Ouchida, and Miss F. Kasahara for the measurement of physicochemical data. REFERENCES I Kanzaki, T., Nakatsui, I., Kitano, K., Sugiyama, Y., Nishio, M. and Ishikawa, M. (1973) Agric. Biol. Chem. 37, 1407-1416 2 Sugiyama, Y., Kitano, K. and Kanzaki, T. (1973) Agric. Biol. Chem. 37, 1607-1612 3 Sugiyama, Y., Kitano, K. and Kanzaki, T. (1973) Agric. Biol. Chem. 37, 1837-1847 4 Dunphy, P. J., Gutnick, D. L., Phillips, P. G. and Brodie, A. F. (1968) J. Biol. Chem. 243, 398407 Fujita, M., Ishikawa, S. and Shimazono, N. (1966) J. Biochem. Tokyo 59, 104-114 2 Kanzaki, T., Isobe, K., Okazaki, H., Motizuki, K. and Fukuda, H. (1967) Agric. Biol. Chem. 31, 13o7-r313 7 Dunphy, P. J. and Brodie, A. F. (1971) in Methods in Enzymology (McCormick, D. B. and Wright, L. D., eds), Vol. XVIIIC, pp. 407-461, Academic Press, New York 8 Beau, S., Azerad, R. and Lederer, E. (1966) Bull Sot. Chim. Biol. 48, 569-581 9 Campbell, I. M. and Bentley, R. (1968) Biochemistry 7, 3323-3327 IO Linn, B. O., Page, Jr, A. C., Wong, E. L., Gale, P. H., Shunk, C. H. and Folkers, K. (1959) J. Am. Chem. Sot. 81, 4007-4010 II Bishop, D. H. L., Pandya, K. P. and King, H. K. (1962) Biochem. J. 83, 606-614 _ __ _ - . - __ . . 12 GaIe, P. H., Arison, B. H., TreMer, N. R., Page, Jr, A. C. and Folkers, K. (1963) Biochemistry 2, 2oo-203 I3 Jeffries, L., Cawthome, M. A., Harris, M., Diplock, A. T., Green, J. and Price, S. A. (1967) Nature 215, 257-259 14 Phillips, P. G., Dunphy, P. J., Servis, K. L. and Brodie, A. F. (1969) Biochemistry 8,2856-2861 I5 Scholes, P. B. and King, H. K. (1965) Biochem. 5. 97, 766-768