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Physiological properties of phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase from Rhodopseudomonas sp. No. 7
T. Inui, M. Anpo, K. Izui, S. Yanagida, T. Yamaguchi (Editors) Advances in Chemical Conversions for Mitigating Carbon Dioxide Studies in Surface Science and Catalysis, Vol. 114 9 1998 Elsevier Science B.V. All rights reserved.
463
P h y s i o l o g i c a l p r o p e r t i e s of p h o s p h o e n o l p y r u v a t e carboxylase and p h o s p h o e n o l p y r u v a t e c a r b o x y k i n a s e f r o m Rhodopseudomonas sp. No. 7 Takaaki Fujii, Megumi Sadaie, M a s a a k i Saijou, Takanari Nagano, Tomoaki Suzuki, Masahiro Ohtani and Hirofumi Shinoyama Department of Bioproduction Science, Faculty of Horticulture, Chiba University, 648 Matsudo, Matsudo-shi, Chiba 271, J a p a n 1.
INTRODUCTION
Purple nonsulfur bacteria absolutely require a CO2 source such as bicarbonate for growth when they are incubated in a medium containing ethanol under photoanaerobic conditions. On the contrary, their growth on acetate is independent of bicarbonate. These facts suggest that the Calvin Benson cycle operates in cells growing on ethanol, but not in cells growing on acetate [1-3~ . Therefore, it is expected that the carbon metabolism in the ethanol culture of the purple nonsulfur bacteria is more intricate than that of the acetate culture although both ethanol and acetate are metabolized via acetyl CoA. However, information on the regulation between the Embden Meyerhof Parnas pathway-Calvin Benson cycle side and the TCA cycle-glyoxylic acid cycle side in purple nonsulfur bacteria is limited [4-8~ . Activity-levels of phosphoenolpyruvate carboxylase (EC 4.1.1.31, PEPC) and phosphoenolpyruvate carboxykinase (EC 4.1.1.49, PEPK) were examined with Rhodopseudomonas sp. No.7 grown photoanaerobically in an ethanol-bicarbonate and in an acetate medium. PEPC and PEPK were purified from cells grown under these conditions, and several characteristics of the enzymes were discussed in connection with photoheterotrophy of purple nonsulfur bacteria. 2.
METHODS AND MATERIALS
2.1. O r g a n i s m , m e d i u m , a n d g r o w t h No.7 and its maintenance were described containing both 0.1% ethanol and 0.2% acetate was used for the main culture. screw-capped 1.5 liter Roux bottles filled conditions.
conditions. Rhodopseudomonassp. previously [2~ . A basal salt medium sodium bicarbonate or 0.27% sodium The organism was grown at 30~ in with the medium under photoanaerobic
2.2. E n z y m e a s s a y s . The activity of PEPC was assayed by a coupled system in which the oxaloacetate formed was reduced to malate by NADH [8~ . The activity of PEPK was assayed principally according to the method for the activity of PEPC except for the addition of ADP and aspartate.
464
2.3. P u r i f i c a t i o n of P E P C a n d P E P K . PEPC was purified by the method described previously [8~ . PEPK was purified principally according to the method for the purification of PEPC. The enzymes were purified as chromatographically, electrophoretically and isoelectrophoretically homogeneous proteins. 3.
RESULTS
3.1. P E P C a n d P E P K a c t i v i t i e s i n R h o d o p s e u d o m o n a s sp. No.7 grown on e t h a n o l or a c e t a t e . The maximum growth of Rhodopseudomonas sp.No.7 a t t a i n e d in the 0.1% (w/v) ethanol medium, when sodium bicarbonate was added to the medium to give the concentration of 0.2 %. On the other hand, the growth in the acetate medium was independent of bicarbonate. When the organism was incubated in a medium containing 0.27 % sodium acetate which was almost equivalent to the carbon m a s s of the 0.1% ethanol-0.2% bicarbonate medium, the maximum growth attained in the acetate medium was similar to t h a t of the ethanol medium. The activity of PEPC in the ethanol-grown cells showed about 3 times higher level t h a n t h a t of the acetate-grown cells (Table 1). There was a significant difference between the activity ratio of PEPC to PEPK in the ethanolgrown cells (11.6) and t h a t in the acetate-grown cells (2.76). Table 1 Effect of c a r b o n sources on activity levels of P E P C a n d P E P K in sp. No.7
Rhodopseudomonas
Carbon source for growth
Specific activity (nmol/min/mg)
Rate of activity (PEPC/PEPK)
PEPC
PEPK
Ethanol + bicarbonate
42.4
3.66
11.60
Acetate
14.1
5.11
2.76
Reprinted from: M. Sadaie,T. Nagano,T. Suzuki,H. Shinoyama,and T. Fujii, Biosci. Biotech. Biochem., 61,625(1997). 3.2. P r o p e r t i e s of P E P C . The molecular weights of the purified enzyme and its subunit were e s t i m a t e d to be about 400,000 and about 102,000, respectively. The optimum pH and t e m p e r a t u r e of the activity were about 9.0 and 50~ respectively. The p / o f the enzyme was about 5.8. The enzyme required Mg 2§ or Mn 2§ for appearance of the activity. The enzyme activity in the presence of 2 mM Mn 2§ was about 25 times higher t h a n t h a t of 2 mM Mg 2§ But an allosteric effector, acetyl CoA, acted on the enzyme more effectively in the presence of Mg 2§ t h a n Mn 2§ (Figure 1). When the enzyme was incubated with 20 mM Mg 2§ the Kms against bicarbonate and PEP decreased to about 1/2 (1.7 mM) and 1/20 (0.23 mM) in the presence of 40 tt M
465 acetyl CoA, respectively, and the Vmax increased to about 30 times (90 tt mol/mirgmg protein). The enzyme was inhibited by aspartate (/~, 0.208 mM). Besides, the enzyme of strain No.7 was strongly inhibited by ATP and GTP (Table 2). The enzyme was also inhibited by ADP. This inhibition was completely reversible. The enzyme activity was hardly affected by various concentrations (0-10 mM) of fructose- 1,6-bisphosphate (FBP).
Table 2 Effect of various nucleotides on PEPC and PEPK activity Nucleotides (4mM) None ATP ADP GTP GDP
Relative activity (%) PEPC 100 4.8 35 12 94
PEPK 0
100 0 0 0
3.3. P r o p e r t i e s of P E P K . The molecular weights of the purified enzyme and its subunit were estimated to be about 120,000 and about 60,000, respectively. The optimum pH and temperature of the activity were about 6.0 and 50 to 60~ respectively. The p/of the enzyme was about 5.7. PEPK absolutely required Mn 2§ for the appearance of its activity. Although the activity of PEPC was reversibly inhibited by ATP(ADP), the activity of PEPK appears only in the presence of ATP(ADP) (Table 2). The activity was not affected by acetyl CoA and aspartate. The decarboxylation activity of PEPK was about three times higher than the carboxylation activity (Figure 2). The Kms against oxaloacetate and ATP were calculated to be 0.31 mM and 0.38 mM in the decarboxylation reaction, respectively. The Vmaxwas 82 tt mol/min/mg protein.
100/ ~o
.
60,]:
[]
___--~
f
0
~100
v .~
60 9
20
40 20
oI
I
10
I
I
I
20 30 40 Acetyl CoA (,aM)
50
Figure 1 Effect of Mg 2+ or Mn 2+ on activation of PEPC by acetyl CoA (9
2raM Mg 2+
(m), 20raM Mg 2+
(0), 0.5raM Mn 2+ (o), 2raM Mn 2+
o
1 2 Enzyme conc. (tz g/ml)
3
Figure 2 Decarboxylation and carboxylation activity of PEPK (O), Carboxylation activity ( 0 ) , Decarboxylation activity
466
4.
DISCUSSION
PEPC and PEPK, which connect the EMP pathway-Calvin Benson cycle side and the TCA cycle-glyoxylic acid cycle side, were detected in the cell-free extracts of Rhodopseudomonas No.7 grown on C2-compounds. There was a significant difference between the ethanol-grown and acetate-grown cells in respect to levels of PEPC and PEPK. The ethanol-grown cells had a higher level of PEPC than that of the acetate-grown cells. The difference of the carbon assimilation observed between the ethanol culture and the acetate culture of strain No.7 has not been reported in nonphoto-heterotrophic microorganisms. PEPCs from enterobacteria such as Salmonella typhimurium and Escherichia coli are activated by acetyl CoA and inhibited by aspartate. Some properties of the enzyme from Rhodopseudomonas sp. No.7 were similar to those of the enterobacteria. However, the strain No.7 enzyme was different from enzymes of the enterobacteria in important points of metabolic regulation. It have been reported that the enzyme activities of the enterobacteria were increased by FBP and GTP. In contrast with this, the activity of the No.7 enzyme was not affected by FBP and reversibly inhibited by GTP and ATP (ADP). This differences may signify roles of the enzyme of strain No.7 related to the ethanol-assimilation and the enzymes of the enterobacteria to the glucose-assimilation in their own way. It is interesting in connection with the assimilation of ethanol and acetate by purple nonsulfur bacteria that the activity of PEPC from Rhodopseudomonas sp. No.7 was reversibly inhibited by ATP(ADP), but the activity of PEPK from strain No.7 appears only in the presence of ATP(ADP). Besides, it was found that the decarboxylation activity of PEPK was higher than the carboxylation activity. Although further investigation is necessary to clarify whether regulatory properties of PEPC and PEPK from strain No.7 are closely related to photoheterotrophic physiology or not, these enzyme seems to play significant roles in the photoheterotrophic growth of strain No.7 on ethanol and acetate. REFERENCES 1. G.A.Sojka, in "The Photosynthetic Bacteria," ed. by R.K.Clayton and W.R.Sistrom, Plenum Press, New York, 1978, pp. 707-718. 2. T.Fujii, A.Nakazawa, N.Sumi, H.Tani, A.Ando and M.Yabuki, Agric. Biol. Chem., 47, 2747 (1983). 3. K.Isida, H.Shinoyama and T.Fujii, Seibutsukougaku, 71, 397 (1993). 4. J.Payne and J.G.Morris, J. Gen. Microbiol.,59, 97 (1969). 5. H.Albers and G.Gottschalk, Arch. Microbiol., 111, 45 (1976). 6. R.C.Fuller, in "The Photosynthetic Bacteria," ed. by R.K.Clayton and W. R. Sistrom, Plenum Press, New York, 1978, pp. 691. 7. J.C.Willison, J. Gen. Microbiol., 134, 2429 (1988). 8. M.Sadaie, T.Nagano, T.Suzuki, H.Shinoyama and T.Fujii, Biosci. Biotech. Biochem., 61, 625 (1997).
463
P h y s i o l o g i c a l p r o p e r t i e s of p h o s p h o e n o l p y r u v a t e carboxylase and p h o s p h o e n o l p y r u v a t e c a r b o x y k i n a s e f r o m Rhodopseudomonas sp. No. 7 Takaaki Fujii, Megumi Sadaie, M a s a a k i Saijou, Takanari Nagano, Tomoaki Suzuki, Masahiro Ohtani and Hirofumi Shinoyama Department of Bioproduction Science, Faculty of Horticulture, Chiba University, 648 Matsudo, Matsudo-shi, Chiba 271, J a p a n 1.
INTRODUCTION
Purple nonsulfur bacteria absolutely require a CO2 source such as bicarbonate for growth when they are incubated in a medium containing ethanol under photoanaerobic conditions. On the contrary, their growth on acetate is independent of bicarbonate. These facts suggest that the Calvin Benson cycle operates in cells growing on ethanol, but not in cells growing on acetate [1-3~ . Therefore, it is expected that the carbon metabolism in the ethanol culture of the purple nonsulfur bacteria is more intricate than that of the acetate culture although both ethanol and acetate are metabolized via acetyl CoA. However, information on the regulation between the Embden Meyerhof Parnas pathway-Calvin Benson cycle side and the TCA cycle-glyoxylic acid cycle side in purple nonsulfur bacteria is limited [4-8~ . Activity-levels of phosphoenolpyruvate carboxylase (EC 4.1.1.31, PEPC) and phosphoenolpyruvate carboxykinase (EC 4.1.1.49, PEPK) were examined with Rhodopseudomonas sp. No.7 grown photoanaerobically in an ethanol-bicarbonate and in an acetate medium. PEPC and PEPK were purified from cells grown under these conditions, and several characteristics of the enzymes were discussed in connection with photoheterotrophy of purple nonsulfur bacteria. 2.
METHODS AND MATERIALS
2.1. O r g a n i s m , m e d i u m , a n d g r o w t h No.7 and its maintenance were described containing both 0.1% ethanol and 0.2% acetate was used for the main culture. screw-capped 1.5 liter Roux bottles filled conditions.
conditions. Rhodopseudomonassp. previously [2~ . A basal salt medium sodium bicarbonate or 0.27% sodium The organism was grown at 30~ in with the medium under photoanaerobic
2.2. E n z y m e a s s a y s . The activity of PEPC was assayed by a coupled system in which the oxaloacetate formed was reduced to malate by NADH [8~ . The activity of PEPK was assayed principally according to the method for the activity of PEPC except for the addition of ADP and aspartate.
464
2.3. P u r i f i c a t i o n of P E P C a n d P E P K . PEPC was purified by the method described previously [8~ . PEPK was purified principally according to the method for the purification of PEPC. The enzymes were purified as chromatographically, electrophoretically and isoelectrophoretically homogeneous proteins. 3.
RESULTS
3.1. P E P C a n d P E P K a c t i v i t i e s i n R h o d o p s e u d o m o n a s sp. No.7 grown on e t h a n o l or a c e t a t e . The maximum growth of Rhodopseudomonas sp.No.7 a t t a i n e d in the 0.1% (w/v) ethanol medium, when sodium bicarbonate was added to the medium to give the concentration of 0.2 %. On the other hand, the growth in the acetate medium was independent of bicarbonate. When the organism was incubated in a medium containing 0.27 % sodium acetate which was almost equivalent to the carbon m a s s of the 0.1% ethanol-0.2% bicarbonate medium, the maximum growth attained in the acetate medium was similar to t h a t of the ethanol medium. The activity of PEPC in the ethanol-grown cells showed about 3 times higher level t h a n t h a t of the acetate-grown cells (Table 1). There was a significant difference between the activity ratio of PEPC to PEPK in the ethanolgrown cells (11.6) and t h a t in the acetate-grown cells (2.76). Table 1 Effect of c a r b o n sources on activity levels of P E P C a n d P E P K in sp. No.7
Rhodopseudomonas
Carbon source for growth
Specific activity (nmol/min/mg)
Rate of activity (PEPC/PEPK)
PEPC
PEPK
Ethanol + bicarbonate
42.4
3.66
11.60
Acetate
14.1
5.11
2.76
Reprinted from: M. Sadaie,T. Nagano,T. Suzuki,H. Shinoyama,and T. Fujii, Biosci. Biotech. Biochem., 61,625(1997). 3.2. P r o p e r t i e s of P E P C . The molecular weights of the purified enzyme and its subunit were e s t i m a t e d to be about 400,000 and about 102,000, respectively. The optimum pH and t e m p e r a t u r e of the activity were about 9.0 and 50~ respectively. The p / o f the enzyme was about 5.8. The enzyme required Mg 2§ or Mn 2§ for appearance of the activity. The enzyme activity in the presence of 2 mM Mn 2§ was about 25 times higher t h a n t h a t of 2 mM Mg 2§ But an allosteric effector, acetyl CoA, acted on the enzyme more effectively in the presence of Mg 2§ t h a n Mn 2§ (Figure 1). When the enzyme was incubated with 20 mM Mg 2§ the Kms against bicarbonate and PEP decreased to about 1/2 (1.7 mM) and 1/20 (0.23 mM) in the presence of 40 tt M
465 acetyl CoA, respectively, and the Vmax increased to about 30 times (90 tt mol/mirgmg protein). The enzyme was inhibited by aspartate (/~, 0.208 mM). Besides, the enzyme of strain No.7 was strongly inhibited by ATP and GTP (Table 2). The enzyme was also inhibited by ADP. This inhibition was completely reversible. The enzyme activity was hardly affected by various concentrations (0-10 mM) of fructose- 1,6-bisphosphate (FBP).
Table 2 Effect of various nucleotides on PEPC and PEPK activity Nucleotides (4mM) None ATP ADP GTP GDP
Relative activity (%) PEPC 100 4.8 35 12 94
PEPK 0
100 0 0 0
3.3. P r o p e r t i e s of P E P K . The molecular weights of the purified enzyme and its subunit were estimated to be about 120,000 and about 60,000, respectively. The optimum pH and temperature of the activity were about 6.0 and 50 to 60~ respectively. The p/of the enzyme was about 5.7. PEPK absolutely required Mn 2§ for the appearance of its activity. Although the activity of PEPC was reversibly inhibited by ATP(ADP), the activity of PEPK appears only in the presence of ATP(ADP) (Table 2). The activity was not affected by acetyl CoA and aspartate. The decarboxylation activity of PEPK was about three times higher than the carboxylation activity (Figure 2). The Kms against oxaloacetate and ATP were calculated to be 0.31 mM and 0.38 mM in the decarboxylation reaction, respectively. The Vmaxwas 82 tt mol/min/mg protein.
100/ ~o
.
60,]:
[]
___--~
f
0
~100
v .~
60 9
20
40 20
oI
I
10
I
I
I
20 30 40 Acetyl CoA (,aM)
50
Figure 1 Effect of Mg 2+ or Mn 2+ on activation of PEPC by acetyl CoA (9
2raM Mg 2+
(m), 20raM Mg 2+
(0), 0.5raM Mn 2+ (o), 2raM Mn 2+
o
1 2 Enzyme conc. (tz g/ml)
3
Figure 2 Decarboxylation and carboxylation activity of PEPK (O), Carboxylation activity ( 0 ) , Decarboxylation activity
466
4.
DISCUSSION
PEPC and PEPK, which connect the EMP pathway-Calvin Benson cycle side and the TCA cycle-glyoxylic acid cycle side, were detected in the cell-free extracts of Rhodopseudomonas No.7 grown on C2-compounds. There was a significant difference between the ethanol-grown and acetate-grown cells in respect to levels of PEPC and PEPK. The ethanol-grown cells had a higher level of PEPC than that of the acetate-grown cells. The difference of the carbon assimilation observed between the ethanol culture and the acetate culture of strain No.7 has not been reported in nonphoto-heterotrophic microorganisms. PEPCs from enterobacteria such as Salmonella typhimurium and Escherichia coli are activated by acetyl CoA and inhibited by aspartate. Some properties of the enzyme from Rhodopseudomonas sp. No.7 were similar to those of the enterobacteria. However, the strain No.7 enzyme was different from enzymes of the enterobacteria in important points of metabolic regulation. It have been reported that the enzyme activities of the enterobacteria were increased by FBP and GTP. In contrast with this, the activity of the No.7 enzyme was not affected by FBP and reversibly inhibited by GTP and ATP (ADP). This differences may signify roles of the enzyme of strain No.7 related to the ethanol-assimilation and the enzymes of the enterobacteria to the glucose-assimilation in their own way. It is interesting in connection with the assimilation of ethanol and acetate by purple nonsulfur bacteria that the activity of PEPC from Rhodopseudomonas sp. No.7 was reversibly inhibited by ATP(ADP), but the activity of PEPK from strain No.7 appears only in the presence of ATP(ADP). Besides, it was found that the decarboxylation activity of PEPK was higher than the carboxylation activity. Although further investigation is necessary to clarify whether regulatory properties of PEPC and PEPK from strain No.7 are closely related to photoheterotrophic physiology or not, these enzyme seems to play significant roles in the photoheterotrophic growth of strain No.7 on ethanol and acetate. REFERENCES 1. G.A.Sojka, in "The Photosynthetic Bacteria," ed. by R.K.Clayton and W.R.Sistrom, Plenum Press, New York, 1978, pp. 707-718. 2. T.Fujii, A.Nakazawa, N.Sumi, H.Tani, A.Ando and M.Yabuki, Agric. Biol. Chem., 47, 2747 (1983). 3. K.Isida, H.Shinoyama and T.Fujii, Seibutsukougaku, 71, 397 (1993). 4. J.Payne and J.G.Morris, J. Gen. Microbiol.,59, 97 (1969). 5. H.Albers and G.Gottschalk, Arch. Microbiol., 111, 45 (1976). 6. R.C.Fuller, in "The Photosynthetic Bacteria," ed. by R.K.Clayton and W. R. Sistrom, Plenum Press, New York, 1978, pp. 691. 7. J.C.Willison, J. Gen. Microbiol., 134, 2429 (1988). 8. M.Sadaie, T.Nagano, T.Suzuki, H.Shinoyama and T.Fujii, Biosci. Biotech. Biochem., 61, 625 (1997).