Abstract. 1. Citrate !yase (EC 4.1.3.6) from Rhodopseudomonas gelatinosa has been purifed to homogeneity by protamine sulfate fractionation, chromatography.
Arch. Microbiol. 100, 307--328 (1974) 9 by Springer-Verlag 1974
Citrate Lyase from Rhodopseudomonasgelatinosa: Purification, Electron Microscopy and Subunit Structure N o r b e r t Beuscher, F r a n k Mayer, a n d Gerhard G o t t s c h a l k Institut ffir ~ikrobiologie der Universit/it G6ttingen und der Gesellschaft fiir Strahlen- und Umwe!tforsehung mbH in G6ttingen Received November 7, 1974
Abstract. 1. Citrate !yase (EC 4.1.3.6) from Rhodopseudomonas gelatinosa has been purifed to homogeneity by protamine sulfate fractionation, chromatography on DEAE-Ce!lulose and gel filtration. The final enzyme preparation had a specific activity of 138 units per mg of protein and was purified 43-fold over the crude extract. Analysis of citrate lyase by sedimentation equilibrium experiments and gel filtration gave molecular weights of 530000 and 560000, respectively. 2. Electron microscopic investigations of negatively stained enzyme molecules and image analysis showed that citrate lyase is composed of six large and six small subunits; they are arranged in two hexagonal rings lying face to face, each containing, in alternating sequence, three large and three small subunits. The enzyme molecule is 160 A in diameter and about !00 A thick. 3. Treatment with sodium dodecylsu!fate and m_ercaptoethanol dissociated citrate lyase into three proteins. Protein I I I (small subunit) had a molecular weight of 30000 and contained the pantothenate; protein II (large subunit) had a molecular weight of 61000; protein I (Mr ~ 97000) was probably an aggregate of IX and IIl. 4. Based
on the results obtained a model
of citrate lyase was constructed.
5. Purified citrate lyase was obtained from R. gelatinosa in a deacetylated and largely oxidized form. The enzyme was activated by reduction with dithiothreitol (3 raM) and subsequent aeetylation with acetic anhydride (1.75 raM). 6. The enzyme was subject to reaction inactivation, the extent of which depended on the concentration of Mg2+.
Key words: Citrate Lyase -- Subunit Structure -- Electron Microscopy -Reaction Inactivation -- Rhodopseudomonas gelatinosa -- Phototrophic Bacteria. Rhodopseudomonas gelatinosa grows p h o t o t r o p h i c a l l y with citrate a n d is able to degrade this c o m p o u n d very r a p i d l y (Weekesser et al., 1969; Schaab et al., 1972). T h e r e b y m o s t of the c i t r a t e - c a r b o n is excreted i n the form of acetate which is utilized for growth after all the citrate of the culture m e d i u m has been consumed. Citrate lyase serves as a key e n z y m e in the d e g r a d a t i o n of citrate b y R. gelatinosa. Following the e x h a u s t i o n of the citrate of the m e d i u m this e n z y m e is r a p i d l y i n a c t i v a t e d b y d e a c e t y l a t i o n (Giffhorn et al., 1972). I n
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o r d e r to s t u d y t h e r e g u l a t i o n o f t h i s i n a c t i v a t i o n r e a c t i o n a p r o c e d u r e for t h e p u r i f i c a t i o n o f c i t r a t e l y a s e h a d to be w o r k e d o u t a n d t h e p r o p e r t i e s o f t h i s e n z y m e h a d t o be i n v e s t i g a t e d . T h e r e s u l t s d e m o n s t r a t e differences in t h e s t r u c t u r e o f t h i s e n z y m e as c o m p a r e d t o t h e c i t r a t e l y a s e f r o m
Klebsiella aerogenes.
Methods Growth o/Bacteria. Rhodopseudomonas gelatinosa strain SMG 149 was grown in 20 1 carboys anaerobically in the light. The medium and the growth conditions were the same as described earlier (Schaab et al., 1972). In order to obtain higher growth yields, 1.5 1 of the ten-fold concentrated medium was added after 24 hrs of growth and the culture was incubated for another 12 hrs. From one carboy usually 38 g of cells (wet weight) were obtained. Since storage of the cell paste caused irreversible loss of enzyme activity the cells harvested were immediately used for the purification of citrate lyase. Determination o] Protein and Ultra/iltration o] Protein Solutions. Protein was determined according to Lowry et al. (1951) with crystalline bovine serum albumin as standard. Pigments were removed by extraction with acetone (2.5 ml per 0.1 ml of protein solution). Protein solutions were concentrated in Diaflo-UF-cells or with the system TCF 10 (Amicon, N.V.). The membranes were those of the PM and XM series with exclusion limits from 10000 to 300000 daltons. Filtration was performed under a nitrogen pressure of 0.7--3.0 atm (depending on the type of membrane) at 4~ The filtration was terminated using the endpoint controller EC 20 (Amicon, N.V.). Standard Reactivation o/ Citrate Lyase and Enzyme Assay. Since citrate lyase preparations were always largely deacetylated and contained considerable quantities of oxidized enzyme, its activity could only be determined following the reduction and the acetylation of the enzyme, l~eactivation of citrate lyase from the S-S- to the SH-enzyme was performed in glass tubes under a nitrogen atmosphere. To 1 ml of enzyme solution containing approx. 1 mg of purified lyase or 2--9 mg of crude enzyme dithiothreitol (DTT) or dithioerythritol (DTE) was added to a final concentration of 3 mM. This mixture was incubated for 20 min at 30 ~C. To determine enzyme activity aliquots (10 ~l) were withdrawn and transferred to 3 ml cuvettes which contained all the components of the assay except citrate. The assay mixture used was a modification of the one described by Dagley (1969) ; it contained in a final volume of 3 ml: 100 mM potassium phosphate buffer, p H 7.2; 3 mM MgCl2; 0.15 mM N A D H ; 24 U of malate dehydrogenase and 14 U of lactate dehydrogenase. Following the addition of reduced enzyme, acetic anhydride or acetyl imidazole was added to final concentrations of 1.75 mM and 10 raM, respectively (Buckel et al., 1971). After 1 rain the reaction was started with citrate (2 mM). One enzyme unit catalyzed the cleavage of 1 izmole of citrate per minute at 30~ Sometimes it was necessary to acetylate several milligrams of lyase. This was carried out by adding acetic anhydride (3.5 raM) or acetyl imidazole (50 mM) to enzyme solutions with a protein content of 1 mg/ml. I f necessary reactivated lyase was freed from the products of the reduction and acetylation reactions by gel filtration with Sephadex G-25. EIectrophoresis. Polyacrylamide-gel electrophoresis was performed in a cell for vertical slabs (Laborger~te Miiller, Hann.-Miinden) at a current of 15 mA for 5 hrs. Agarose-polyacrylamide gels (0.5 ~- 3~ according to Peacock and Dingman (1968) or 5O/o polyacrylamide gels with 4 mm thickness were used. Disc-electrophoresis with polyacrylamide gels in glass tube was conducted in an apparatus from W T W
Citrate Lyase from R. gelatinosa
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(Munich) using gel systems with 3.750/0 and 50/0 acrylamide according to Maurer (1968). Sodium dodecylsulfate electrophorcsis was performed in the above mentioned a p p a r a t u s for vertical slab clectrophoresis (10~ gels) according to Weber and Osborn (1969). S t a n d a r d proteins and the purified enzyme in 25 mM sodium phosp h a t e buffer, p H 7.1 were incubated with 1~ SDS a n d 1~ 2-mercaptoethanol ( • 8 M urea) for 2 min a t 100~ Then the solutions were cooled down to 30~ and dialyzed against 0.1 ~ SDS ~- 0.1 ~ 2-mercaptoethanol for 6 hrs. Variations of this s t a n d a r d denaturation procedure for controlling a reproducible dissociation were t a k e n from Weber et al. (1972). D e n a t u r a t e d proteins were mixed with sucrose plus some crystals of bromphenol-blue and applied to the gels. EIectrophoresis was carried o u t in the above mentioned buffer + 0.1~ SDS a t a current of 80 mA for approximately 5 hrs. The slabs were stained and destained according to Koenig et al. (1970). Sometimes the prestaining procedure of Griffith (1972) was used. Determination o] Molecular Weight. Analytical uitracentrifugation experiments were performed in a Beckman-Spinco model E ultracentrifuge equipped with monochromator, multiplexer and photoeleetrical scanner. Double-sector cells with sapphire-windows and 12 m m tight-path were used. Electron Microscopy. For electron microscopy citrate lyase solutions in 10 mM potassium phosphate buffer, p H 7.2 with a protein content of 50--100 ~g/ml were used. The enzyme was prepared for electron microscopy by a negative staining procedure based on the one reported b y Valentine et al. (1968) b u t modified as follows: The negative stain was prepared b y dissolving 2 g of uranyl acetate in 50 ml of water. This negative staining solution had a pH of 5 and was used without dilution. The enzyme molecules were dissolved either in phosphate buffer as described above or fixed for a b o u t 20 min in the same buffer containing 0.50/0 glutaraldehyde. They were applied to a carbon film deposited on cleaved mica by floating the film off on the surface of the enzyme solution. After 10 to 60 sec, t h e carbon film was transferred to a water surface where it was allowed to float for 2 to 3 sec. Thus, the salts of the enzyme solution could diffuse into the fluid layer adjacent to the carbon film. Finally, the carbon film with the adsorbed and washed enzyme molecules was transferred to a container (beem capsule) with negative stain where it was left floating for 20 to 120 see. During this time, the water surrounding the enzyme molecules was replaced by negative staining solution. The floating carbon film was picked up with a carbon coated copper grid immersed in the negative staining solution with the carbon side showing up. The carbon coated copper grid was lifted out of the negative staining solution together with the floating carbon film containing the adsorbed enzyme molecules. Thus, after blotting the grid on filter paper, negative stain a n d enzyme molecules were captured between two carbon layers forming a "sandwich". The carbon coated copper grids used for the pick-up procedure were not completely covered b y e~rbon. Therefore, there were regions in t h e final preparation where the picked-up carbon film with adsorbed enzyme molecules was not covered b y a second carbon film. Electron microscopy was performed using the electron microscopes EM 9 S-2 (Zeiss, Oberkochen), J E M 100 B (JEOL, J a p a n ) and EM 301 (Philips, Eindhoven) at primary magnifications of 25000 • to 90000 X.
Microbiological Determination o] the Pantothenate Content o/ Citrate Lyase. Citrate lyase and coenzyme A as reference compound were hydrolyzed following the procedure described b y P u g h a n d Wakil (1965). The enzyme was first precipitated with 3 M trichloroacetic acid and t h e n dissolved in 5 ml of 1 M KOH. Coenzyme A was also dissolved in 1 M KOH. Both solutions were transferred to ampoules which subsequently were sealed and incubated a t 100~ for 2 hrs. Then the p H was adjusted to 8.5 with 5 M HC1, and Tris-HC1 buffer, p H 8.5 was added to a final con-
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centration of 0.1 M. Precipitates formed were removed by centrifugation (10 rain at 5000 • g). The pellet was washed once with 2 ml of water, and the washing was combined with the first supernatant. Alkaline phosphatase was added (10 ~g/ml), and the solutions were incubated at 37~ for 24 hrs. Pantothenate was determined with Lactobacillus plantarum (DSM 20 205) following the procedure of Difco Laboratories (1971). Enzymes and Chemicals. Coenzyme A, NADH, malate dehydrogenase, lactate dehydrogenase and the reference proteins for molecular weight determinations were obtained from Boehringer Mannheim GmbH (Mannheim, Germany). Urease and glutamate dehydrogenase were purchased from Miles-Servac, London. The chemicals for gel electrophoresis, 5,5'-dithiobis (2-nitrobenzoic acid) and protamine sulfate were obtained from Serva, Heidelberg. Results
Purification of Citrate Lyase from Rhodopseudomonas gelatinosa 250 g of fresh cells from R. gelatinosa were suspended in 850 ml 75 mM potassium phosphate buffer, p H 7.2 containing MgCl~ and DTE in final concentrations of 3 mM and 1 raM, respectively. This mixture was stirred at 4~ overnight under a nitrogen atmosphere. Then 40 ml portions of the suspension were passed quickly through a pre-cooled French press at a pressure of 80 kp/cm 2. The extract was collected in an Erlenmeyerflask which was cooled with ice. Cell debris was removed by centrifugation at 20000 • g for 30 min at 4~ The following steps were carried out within the temperature range of 0 - - 4 ~C. Centri/ugation and Ultra/iltration. Membrane fragments and chromatophores were removed from the extract by ccntrifugation at 100000 • g for 4 hrs. The pellet contained some citrate lyase and was resuspended in 300 ml of the above buffer and centrifuged again (100000 • g, 4 hrs). The combined supernatants were concentrated to 150 ml using the membrane XM 300 which retained molecules with a molecular weigh t greater than 300000 daltons only. Treatment with Protamine Sul/ate. 0.1 volume of a 2 ~ solution (w/v) of protamine sulfate was added dropwise to the concentrated extract with stirring for 30 min. The precipitate was removed by centrifugation at 20 000 • g for 40 min. DEAE-Cellulose Chromatography. The supernatant of the last step was concentrated by ultrafiltration to a volume of 200 ml and allowed to flow into a column (5.0 cm diameter • 45 cm) of DEAE-Cellulose, equilibrated against 75 mM phosphate buffer, p H 7.2 ~ 3 mM MgCl~. The column was washed with the same buffer until the absorption of the eluate was 0.08 at 280 rim. Then the column was developed with 400 ml of 100 mM phosphate buffer, pH 7.2 containing 3 mM MgCl~ ~ 50 mM KC1. Fractions of approximately 6 ml were collected at an average flow rate of 90 ml/hr. The fractions 415--435 were combined and concentrated by ultrafiltration to a volume of 15 ml.
Citrate Lyase from R. gelatinosa
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N u m b e r oI fractions
Fig. 1. Elution of citrate lyase from the first Sepharose 6 B column. Enzyme activity was determined by using 5 ~xl of each fraction reactivated as described in "SIethods". o o protein absorption; o []enzyme activity
Sepharose 6 B Chromatography. The concentrated protein solution of the previous step was applied to a column (2.6 cm diameter • 60 cm) of Sepharose 6 B which had been equilibrated against 100 mM phosphate buffer, p t I 7.2 ~- 3 mM MgCI~. I t was connected to a second column with Sepharose 6 B (2.5 em diameter • 90 em) so t h a t the eluate of the first column passed the second one from the bottom to the top. Both columns were developed with the equilibration buffer. A constant fraction volume of 1.5 ml and a flow-rate of 9 mt/hr were maintained by the use of a Mariott's flask. After concentration of the citrate lyase containing fractions (240--266) to a volume of 4.6 ml the chromatography on Sepharose 6 B was repeated with a column of smaller dimensions (1.5 em diameter • 80 era). The elution of protein and enzyme activity from the columns described is shown in Figs. 1 and 2. The purification procedure is summarized in Table 1. The final preparation with a specific activity of 139 U/mg was stored in 1.0 ml portions at -- 18~ Homogeneity and Molecular Weight. Homogeneity of the purified citrate lyase was examined b y polyaeryl~mide electrophoresis on vertical slabs and by discontinuous gel eleetrophoresis in tubes. I n the experiment described in Fig. 3 the protein solution from the first gel filtration step and the final enzyme preparation were analyzed. Fig.4 shows the densitogram of a stained gel from an analytical disc-eleetrophoresis of the enzyme. Both figures demonstrate t h a t the purified lyase was homogeneous by the criterion of polyacrylamide electrophoresis. The molecular weight of citrate lyase was determined b y two methods: b y low-speed sedimentation equilibrium (van Holde and Baldwin,
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