Purification and some properties of citrate synthase from a nitrite-oxidizing chemoautotroph, Nitrobacter agilis ATCC 14123

JOURNAL OF FEI~ImNTATION A N D BloENam~m~,rG Vol. 77, No. 1, 97-99. 1994

Purification and Some Properties of Citrate Synthase from a Nitrite-Oxidizing Chemoautotroph, Nitrobacter agil& ATCC 14123 REIJI T A K A H A S H I , K A Z U H I T O USUI, TAKASHI SAKURABA, AND TATSUAKI TOKUYAMA*

Department of Agricultural Chemistry, College of Agriculture and Veterinary Medicine, Nihon University, Setagaya, Tokyo 154, Japan Received 8 July 1993/Accepted 18 September 1993

Citrate (si)-synthase (citrate oxaloacetate-lyase, EC 4.1.3.7) was purified as an electrophoretically homogeneous protein from a nitrite-oxidizing chemoautotrophic bacterium, Nitrobacter agilis ATCC 14123. The molecular mass (Mr) of the native enzyme was estimated to be about 250,000 by gel filtration, whereas SDS-PAGE gave two bands with Mr values of 45,000 and 80,000, respectively, suggesting that the enzyme is a tetramer consisting of two different subunits (a: 45,000, ~ 80,000). The isoelectric point of the enzyme was 5.4. The pH and temperature optima on the citrate synthase activity were about 7.5-8.0 and 30-35°C, respectively. The citrate synthase was stable in the pH range of 6.0-9.0 and up to 55°C. The apparent Kn values for oxaloacetate and acetyl-CoA were about 27 pM and 410 pM, respectively. The activity of citrate synthase was not inhibited by ATP (1 mM), N A D H (1 mM) or 2-oxoglutarate (10 mM), but was strongly inhibited by SDS (1 mM). Activation by metal ions was not observed.

Citrate (st)-synthase [EC 4.1.3.7, citrate oxaloacetatelyase] catalyzes the first reaction of the tricarboxylic acid (TCA) cycle and thus plays an important role in cellular metabolism (1). The enzyme catalyses the reaction: AcetylCoA + Oxaloacetate + H20---~Citrate + CoA-SH. Recently, we purified a citrate synthase from the ammonia-oxidizing obligatively chemoautotrophic Nitrosomonas europaea ATCC 25978 (2). Until then, no one had succeeded in obtaining a pure sample of a Nitrobacter citrate synthase, largely due to the poor growth of the strain on conventional media. In this paper, we describe the results on the purification and some properties of citrate synthase from a nitrite-oxidizing facultative chemoautotroph, Nitrobacter agilis, and compare its physical properties with those of the citrate synthase from N. europaea ATCC 25978. N. agilis ATCC 14123 was used throughout this study. The culture medium (BE-medium) was composed of NaNO2, 2.0 g; KH2PO4, 0.15 g; NaCI, 0.5; MgSO4.7H20, 0.05g; CaCO3, 7mg; (NH4)2MoTO24.4H20, 0.05mg; chelated iron (Fe-EDTA, Dojindo Laboratories Co., Kumamoto), 74 mg and deionized water, 1 1 (3). The initial pH of the medium was 7.6-7.8. The seed culture was prepared in a 3-1 Erlenmeyer flask containing 1 1 of the medium by rotary shaking of the culture (200 rpm) for 10 d after inoculation of 100 ml of the stock culture. The main culture was done for 7 d in a 10-1 flat-bottom flask fitted with an aeration apparatus, containing 101 of the medium. All cultivations were performed at 29°C. Enzyme activity of citrate synthase was assayed using the method of Srere et al. (1). The formation of coenzyme A from oxaloacetate plus acetyl-CoA, was measured by following the increase in absorbance at 420 nm with a Shimadzu 160A spectrophotometer equipped with a thermostatted cell compartment, at 35°C. The standard reaction mixture contained, in a total volume of 3 ml, 0.2 mM

oxaloacetate, 0.16 mM acetyl-CoA, 0.1 mM 5,5-dithiobis (2-nitrobenzoic acid), 2 0 m M phosphate buffer (pH 8.0) and enzyme. One unit of citrate synthase was defined as that quantity of the enzyme which catalyzed the formation of one micromole of CoA-SH per minute. Specific activity was expressed as the enzyme units per mg of protein. Protein was determined by the method of Bradford with bovine serum albumin as a standard (4). Protein in column eluates was routinely monitored by the absorbance at 280 nm. Citrate synthase was isolated from cells as follows, all operations being performed at 0--4°C. Wet cells (5.0 g) obtained from 501 of liquid culture were disrupted with a French press (500 kg/cm2). The homogenate was centrifuged at 12,000×g for 30min to remove unbroken cells and cell debris. The supernatant was centrifuged at 140,000 × g for 30 min and the resultant supernatant was used as an active fraction. Solid ammonium sulfate was added to the active fraction. The 40-90% ammonium sulfate precipitate was collected by centrifugation and dissolved in a small volume of 50 mM potassium phosphate buffer, pH 8.0 (PPB), and the solution was dialyzed for 12 h against the same buffer. The dialyzed solution was applied to a column (1.6 × 30 cm) of DEAE Sepharose CL6B (Pharmacia Fine Chemicals, Sweden) equilibrated with PPB. The column was eluted with a linear gradient of 0.0-0.8 M NaC1. Active fractions (40ml) were pooled and concentrated to a volume of 1.0 ml by a Centriprep 30 concentrator (Amicon Division, W.R. Grace & Co., Conn., USA). The enzyme solution was applied on a Sephacryl S-200 HR (Pharmacia) column (1 x 50cm) equilibrated with PPB containing 0.2 M sodium chloride. The elution was done with the same buffer at a flow rate of 0.1 ml/min and the active fractions were pooled. The enzyme solution (6 ml) was concentrated to 0.5 ml by the Centriprep-30 concentrator. The enzyme solution was applied to a Mono Q (10/10) column equilibrated with PPB. The elution of the

* Corresponding author. 97

98

TAKAHASHI ET AL.

J. FERMENT. BIOENG.,

TABLE 1. Summaryof the purification of citrate synthase'from N. agilis Step Cell-free extract Ammonium sulfate ppt (40-90% saturation) DEAE Sepharose CL-6B Sephacryl S-200 HR Mono Q Superose 12HR

Total protein (rag) 514.9 111.4 9.08 5.08 0.302 0.098

enzyme was performed by a linear gradient of sodium chloride (0 to 0.3 M) in the same buffer. The active fractions were collected, and the enzyme was dialyzed against PPB. The enzyme solution (5 ml) was concentrated to 0.1 ml by a Centricon microconcentrator (Amicon). The concentrated enzyme solution was applied on a Superose 12 HR (Pharmacia) column equilibrated with PPB containing 0.1 M sodium chloride. The elution was done with same buffer at a flow rate of 0.2 ml/min; the active fractions were pooled, and the enzyme was dialyzed against PPB. The homogeneity of the enzyme preparation was examined by polyacrylamide slab gel electrophoresis (PAGE) (5). Electrophoresis under nondenaturing conditions was done on PAG Plate 4/15 gels (4-15%) (Dalichi Pure Chemicals Co. Ltd., Tokyo). Gels were run at a constant current of 30mA for 1.5 h. Proteins were stained by the silver stain method (6). The molecular weight of the purified enzyme was estimated by gel filtration with a Superose 6HR (Pharmacia) column according to the method of Andrews (7). The column (I.0 × 30 cm) was equilibrated with PPB containing 0.1 M sodium chloride and eluted at a flow rate of 18ml/h. The eluate was divided into 0.25ml fractions. Apoferritin; horse spleen (Mr, 443,000), /~-amylase (Mr, 200,000) and alcohol dehydrogenase (Mr, 150,000) (Sigma Chemical Co. Ltd., USA) were used as marker proteins. The molecular weight of the purified enzyme was also determined by SDS-polyacrylamide gel electrophoresis (SDSPAGE) as described by Weber and Osborn (8) using

-

Total activity (units) 35.22 33. l0 25.49 22.35 6.9 2.36

Specificactivity (units/mg-protein) 0.07 0.30 2.81 4.40 22.85 24.06

Purification (fold) 1.0 4.3 40.1 62.9 326.4 344.0

Yield (%) 100 94 72.4 63.5 19.6 6.7

SDS-PAG Plate 4/20 (Daiichi) with Tris-glycine buffer, pH 8.4 containing 0.1% SDS. The electrophoresis was performed at 60 mA for 60 min. The marker proteins used were fructose-6-phosphate kinase (Mr, 84,000), serum albumin (Mr, 66,000), fumarase (Mr, 48,500), carbonic anhydrase (Mr, 29,000), /3-1actoglobulin (Mr, 18,400) and alactalbumin (Mr, 14,400) (Sigma). The proteins were detected by the silver stain method. Isoelectric focusing (9) was performed using Ampholine PAG plate (pH 3.5-9.5) (Pharmacia) gels on an Atto AE3230 (Atto Co. Ltd., Tokyo) apparatus. The gels were prefocused at 300 V for 60rain and focused at 300 V for 120 min at 4°C. Anode and cathode solutions were 1.0 M H3PO4 and 1.0 M NaOH, respectively. After electrofocusing, the protein in the gels was stained with silver stain. pI marker proteins with values of 3.5-9.5 were used as references. Results of purification are summarized in Table 1. Citrate synthase was purified 344-fold with a 6.7% activity yield. Specific activity was 24 U/mg. Homogeneity of the purified enzyme solution was examined by PAGE at pH 8.6. In Fig. 1, an electrophoretic pattern of the enzyme is seen, indicating that the preparation is electrophoretically homogeneous. The isoelectric point of the enzyme was 5.4. The molecular weight of the native enzyme was determined to be 250,000 by gel filtration with a Superose 6HR column. The molecular weight of the denatured enzyme was estimated by SDS-PAGE. Two protein bands

100

ori

80

,~ Fructose-6-Phosd~te Kinase ~<"/~

Citratesynthase

g6o sorum~umln Fumar ,_~

40 Carbonic anhydrase

\

0

!

a-Laclalbumlnil 10

FIG. 2.

FIG. 1. Polyacrylamide gel electrophoresis of AT. agilis citrate synthase. Electrophoresis was carried out as described in the text. The arrow indicates the position of the enzymeprotein.

I 0.2

Estimation

I ' = 0.3 0.4 0.5

= 0.6

= 0.7

=

I

0.6

o f molecular weight o f N.

agilL~

citrate

syntbase by SDS-polyacrylamidegel electrophoresis. Symbols: ©, citrate synthase; (a) Mr 45,000, (t3) Mr 80,000. Electrophoresis conditions are described in the text.

VoL. 77, 1994

NOTES

TABLE 2. Properties of citrate synthase from N. agilis Optimum pH Optimum temperature (°C) Heat stability (°C, 10 min) pH stability (5°C, 24 h) pI Molecular weight (M~) Gel filtration SDS-PAGE Km values (/2M) for Acetyl-CoA Oxaloacetic acid

7.5-8.0 30-35 55 6.0-9.0 5.4 250,000 45,000 80,000 410 27

were detected on the gel (data not shown), and the results showed that the enzyme consisted o f two different subunits with molecular weights 45,000 (a) and 80,000 (/9) (Fig. 2). Thus the citrate synthase o f strain A T C C 14123 is a "large" enzyme (ax/3~). Citrate synthases o f Gram-negative bacteria are molecules considerably larger (Mr, 240,000280,000) than those o f Gram-positive bacteria (Mr, 60,000-100,000) (10-13). A large molecular size has also been d e m o n s t r a t e d for the citrate synthase o f N. europaea A T C C 25978 (Mr, 295,000) (2). The effect o f the concentration o f the substrate on the enzyme activity was examined for oxaloacetate and acetyl-CoA. The Michaelis constants for these substrates in citrate synthase were calculate from Lineweaver-Burk plots using the purified enzyme (data not shown). The a p p a r e n t Km values o f the enzyme for oxaloacetate and a c e t y l - C o A were 27/2M and 410/~M, respectively. The Km value for oxaloacetate o f N. agilis enzyme (27 ~M) was essentially the same as that o f the N. europaea enzyme (25/~M). The Km value for a c e t y l - C o A o f N. agilis enzyme (410/~M) was almost the same as that o f Natronbacterium pharaopaea (446 p M ) or Escherichia coli (400/2M) (14), but different f r o m that o f N. europaea (80/2M). The enzyme was most active at p H values ranging between 7.5-8.0 when the enzyme activity was measured under s t a n d a r d assay conditions. The enzyme was stable in the p H range o f 6.0-9.0 when kept at 4°C for 24 h. The enzyme showed the highest activity at 35°C under the s t a n d a r d assay condition. The enzyme was incubated at various temperatures (20-70°C) for 10 min in P P B , and the remaining activity was assayed by the standard assay method. The enzyme was stable at temperatures below 55°C. The effects o f metal ions and inhibitors on the enzyme activity were assayed. Each c o m p o u n d examined was a d d e d to the enzyme, followed by standing for 10min at 35°C, and then the activity was measured. The c o m p o u n d s , SDS, CuSO4 and HgCI2 significantly inhibited the activity at a concentration o f 1.0 m M . The activity o f the enzyme was not significantly inhibited by the c o m p o u n d s E D T A , NAN3, C H 2 I C O O H , K C N and ophenanthroline at a concentration o f 1 mM. The enzymatic and physicochemical properties o f the purified enzyme are summarized in Table 2. In contrast to the enzyme f r o m Gram-positive bacteria, citrate synthases from Gram-negative bacteria are sensitive to feedback inhibition by N A D H (10). The citrate synthase o f the Gram-negative c h e m o a u t o t r o p h Thiobacillus A2 suffers appreciable inhibition due to 1 m M N A D H , but citrate synthases o f T. denitrificans, T. novellus and T. neapolitans are not affected by N A D H (11). The enzyme from Gram-negative N. europaea (2) and N. agilis were

99

also not affected by N A D H (1 mM). W r i g h t et al. (15) f o u n d that 2-oxoglutarate inhibits the citrate synthase f r o m facultatively anaerobic Gram-negative bacteria, f r o m the facultatively anaerobic Gram-negative bacterium E. coli. The citrate synthase o f N. agilis was not inhibited by 2-oxoglutarate (10 m M ) and A T P (1 mM), which is similar to the case o f Nitrosomonas (2). F r o m these results, the citrate synthases from b o t h the facultatively autotrophic N. agilis and obligatively autotrophic N. europaea were shown to have no m a j o r differences in properties except in molecular weight and the Km value for acetyl-CoA. F u r t h e r m o r e , as described above, there are also no significant differences between the properties o f the enzyme from autotrophic and heterotrophic bacteria. The authors are grateful to Dr. H. Sakurai and Mr. T. Ohmori for technical assistance. This work was supported in part by a Grant-in Aid for Scientific Research from Nihon University, Japan. REFERENCES 1. Srere, P.A. and Sanwai, B.D.: Citric acid cycle, p. 3-36. In Grossman, L. and Moldave, K. (ed.), Methods in enzymoiogy, vol. 13. Academic Press, New York (1969). 2. Takahashi, R., Ohmori, T., Kondo, H., and Tokuyama, T.:

3. 4. 5.

6. 7. 8. 9.

10. 11. 12. 13.

Purification and some properties of citrate synthase from ammonia-oxidizing chemoautotrophic Nitrosomonas europaea ATCC 25978. Bulletin of Japanese Society of Microbial Ecology, 7, 4754 (1992). Boek, E. and Engel, H.: Untersuchungen fiber Postoxidative CO2-Fixierung bei Nitrobacter winogradskyi Buch. Arch. Mikrobiol., 57, 191-198 (1966). Bradford, M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72, 248-254 (1976). Haines, B.D.: An introduction to polyacrylamide gel electrophoresis, p. 5--41. In Hames, B. D. and Rickwood, D. (ed.), Gel electrophoresis of proteins. IRL Press, Oxford, Washington DC (1981). Oakey, B.R., Kitsch, D.R., and Morris, N.R.: A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal. Biochem., 105, 361-363 (1980). Andrews, P.: Estimation of the molecular weight of proteins by Sephadex gel-filtration. Biochem. J., 91, 222-223 (1964). Weber, K., Pringle, J.R., and Osborn, M.: Enzyme structure part c, p. 3-27. Hirs, H. W. and Timasheff, S. N. (ed.), Methods in enzymology, vol. 26. Academic Press, New York (1972). Vesterbarg, O. and Svensson, H.: Isoelectric fractionation, analysis, and characterization of ampholytes in natural pH gradients. IV. Further studies on the resolving power in connection with separation of myoglobins. Acta Chem. Scand., 20, 820-834 (1966). Weizman, P. D. J. and Jones, D.: Regulation of citrate synthase and microbial taxonomy. Nature, 219, 270--272 (1968). Weizman, P. D. J. and Dunmore, P.: Regulation of citrate synthase activity by a-ketoglutarate metabolic and taxonomic significance. FEBS Lett., 3, 265-267 (1969). Weizman, P. J. D. and Kinghorn, H. A.: Occurrence of "large" or "small" forms of succinate thiokinase in diverse organisms. FEBS Lett., 88, 255-258 (1978). Weizman, P. J. D.: Unity and diversity in some bacterial citric acid cycle enzymes. Adv. Microb. Physiol., 22, 185-244 (1981).

14. Danson, M.D., Black, S.C., Woodland, D.L., and Wood,

P.A.: Citric acid cycle enzymes of the archaebacteria: citrate synthase and succinate thiokinase. FEBS Lett., 179, 120-124 (1985). 15. Wright, J. A., Maeha, P., and Sanwal, B. D.: Allosteric regulation of the activity of citrate synthase of Escherichia coli by aketoglutarate. Biochem. Biophys. Res. Commun., 29, 34-38 (1967).