J. Biochem. 112, 849-855 (1992)
Purification and Characterization of Monomeric Isocitrate Dehydrogenase with NADP+-Specificity from Vibrio parahaemolyticus Y-4 Noriyuki Fukunaga,1 Sigeki Imagawa,2 Takehiko Sahara, Atsushi Ishii, 3 and Masahiro Suzuki Department of Biology, Faculty of Science, Hokkaido University, Kita-ku, Sapporo, Hokkaido 060 Received for publication, April 15, 1992
NADP+-dependent isocitrate dehydrogenase [IDH: EC 1.1.1.42] was purified to electrophoretic homogeneity from Vibrio parahaemolyticus Y-4, and shown to be a monomeric protein of molecular weight 80,000 with a pi of 5.0. The amino acid composition and partial sequence at the N-terminus resembled those reported for other bacterial monomeric IDHs. Immunotitration with antisera to the monomeric and dimeric enzymes (antisera to EDH-II and -I of Vibrio ABE-1) showed an iTnTniinof.hftmip.nl distinction between the monomeric and dimeric IDHs, but there is similarity within the IDHs of each group. The circular dichroism spectra of the native and heat-denatured enzyme are also similar to those of monomeric IDH (IDH-II of Vibrio ABE-1). These monomeric IDHs are proteins comprising 17-22% helix and 25-35% £-pleated sheet in the native state.
Isocitrate dehydrogenase (IDH) catalyzes the oxidative decarboxylation of isocitrate to 2-oxoglutarate and C0 2 , and is distributed in a wide variety of organisms. In eukaryotic cells, more than two isozymes of IDH differing in their intracellular localizations, structures, and coenzyme specificities are present (2). However, bacterial IDHs from many sources are NADP+-specific (e.g. 2-4) or show much higher affinity to NADP+ than to NAD+ as a cofactor (5, 6), although NAD+-linked IDH is present in a few bacteria (7, 8). In contrast to eukaryotic cells, most bacteria were widely believed to possess either monomeric or dimeric form of IDH, until structurally different IDH isozymes were reported in Acinetobacter calcoaceticus (9) and a psychrophilic marine bacterium, Vibrio sp. strain ABE-1 (Vibrio ABE-1, 10), isolated from sea water off the coast of Hokkaido (11). We previously reported (10) that NADF^-specific IDH-I and -II of Vibrio ABE-1 are a thermostable dimeric form consisting of homologous subunits of MT 46,000 and a thermolabile monomeric form of Mr 80,000, respectively. Furthermore, the N-terminal amino acid sequences of the EDH isozymes (10, 12) are quite different from each other, though that of the IDH-I is similar to that of Escfierichia coli IDH, which is a dimer (23). To our knowledge, coexistence of such different IDH isozymes has not been reported in any other bacterial strain. Recently, Leyland and Kelly (6) reported that the IDH of the photosynthetic bacterium, Rhodomicrvbium vanielii, is a monomer and its N-terminal amino acid sequence is similar to that of the IDH-II. Based on the comparison between amino acid sequences of E. coli EDH and yeast mitochondria! IDH, which is an NAD+-specific octamer consisting of two non-identical subunits, Cupp and Mc1 To whom correspondence should be addressed. Present addresses: * Niigata Research Laboratory, Mitsubishi Gas Co., Niigata 950-31; "Group on Neuropharmacology, Faculty of Pharmacy, Josai University, Sakado, Saitama 350-02.
Vol. 112, No. 6, 1992
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Alister-Henn (14) indicated that one of the subunits of the yeast IDH contains a region similar in sequence to that of the E. coli IDH. These results have implications for the molecular evolution of IDH. The goal of our study is to elucidate the physiological and evolutional meaning of the coexistence of the IDH isozymes in Vibrio ABE-1. Initially, we investigated whether another bacterial strain belonging to genus Vibrio possesses structurally different IDH isozymes like those of Vibrio ABE-1. For this purpose, we purified EDH from Vibrio parahaemolyticus Y-4, because this bacterium is a mesophile with slightly halophilic nature. We found that this bacterium possesses a single EDH of monomeric form with similar N-terminal amino acid sequence, immuno-crossreactivity, and secondary structure to those of EDH-II. MATERIALS AND METHODS Bacteria and Culture Conditions—Vibrio ABE-1 (22) and V. parahaemolyticus Y-4 (V. parahaemolyticus, given to us by Dr. Kazuyuki Kimura of the Department of Microbiology, Faculty of Medicine, Hokkaido University) were cultured with vigorous shaking in a nutrient medium consisting of 1% peptone and meat extract with 0.5 M NaCl at 15 and 37"C, respectively. Azotobacter uinelandii strain LAM 1078 (kindly donated by the Institute of Applied Microbiology, The University of Tokyo) was cultured as described (15). The other bacterial strains used in this study were cultured at the respective optimum temperatures in the nutrient media described above, but 50 mM NaCl was used. Cells in the early stationary phase were harvested and washed three times with chilled 0.5 M NaCl, then frozen at — 80*C until use. Assay of IDH Activity—NADP+-EDH activity was determined spectrophotometrically at 20'C for EDH-EI or at 37°C for V. parahaemolyticus EDH unless otherwise stated. Components of the reaction mixture were the same as
850 described previously (20). The assay system for NAD+IDH was the same as the NADP+-IDH assay system except that NAD+ was used instead of NADP+ and at least 10 times more enzyme protein was employed. One unit of the enzyme activity is defined as the amount of enzyme catalyzing the formation of 1 /*mol of product per min. Protein concentrations were determined by the method of Bradford (16) with bovine y-globulin as a standard. Electrophoresis—Conditions for native and denaturing polyacrylamide gel electrophoresis were the same as described previously (20). Proteins were stained with Coomassie Brilliant Blue R-250. Analytical isoelectric focusing was performed with a slab gel of 5% acrylamide containing 2% Ampholine of pH 3.5-10, and 4 mM isocitrate as described previously (10). Amino Acid Analysis and N-Terminal Sequencing— Purified IDH protein was hydrolyzed in 6 M HC1 at 110'C for 24, 48, and 72 h. The hydrolysates were analyzed with a Hitachi model 835 amino acid analyzer. Cysteine, methionine, and tryptophan were estimated as described previously (10). Amino acid sequencing was performed by automated Edman degradation with an Applied Biosystems 477A gas phase protein sequencer. Immunological Studies—Rabbit antisera and IgG fractions against the IDH-I and -II were obtained by the procedure described previously (10), and frozen at —80'C until use. Immunotitration with each of the above antisera was performed by essentially the same method as described previously (10). For the buffer system, 20 mM potassium phosphate (pH 6.5) containing 0.5 M NaCl, 2 mM MgCl2, 1 mM EDTA, and 10 mM 2-mercaptoethanol was used in the experiments. IDH activity of the crude extract was adjusted to 0.3-0.5 unit/ml, and the antiIDH-I and -II antisera were diluted to 2- and 4-fold, respectively, with the above buffer containing 1% bovine serum albumin. The relative immunocross-reactivity between the purified IDH of V. parahaemolyticus and IDH-II of Vibrio ABE-1 was determined by the method of quantitative, single tube, kinetic-dependent enzyme-linked immunosorbent assay as described (17). Measurement of Circular Dichroism—Circular dichroism (CD) was measured with a Jasco J-20 spectropolarimeter at desired temperatures under constant nitrogen flush. IDH samples (about 0.5 mg protein/ml of 20 mM potassium phosphate buffer, pH 7.0, containing 100 mM NaCl, 5 mM tripotassium citrate, and 2 mM MgCl2) were divided into two portions. Half of each sample was used for CD measurement, and the other half to check the enzyme activity. Prior to the CD measurement, the IDH samples were incubated at the desired temperature for 30 min to attain temperature equilibration. The data were expressed in terms of mean residue eUiptdcity [0]. In order to* estimate secondary structure, we employed a CD spectrum analysis program (28) based on the Chung-Wu-Yang method (29). Chemicals—DEAE-Toyopearl 650M was purchased from Tosoh. Phenyl-Sepharose CL-6B, Red-Sepharose CL-6B, and pi marker proteins were obtained from Pharmacia. Marker proteins for molecular weight were purchased from Boehringer-Mannheim. NAD+ and NADP+ were the products of Oriental Yeast. Ampholine, o-dianisidine, and goat anti-rabbit-IgG-peroxidase conjugate were
N. Fukunaga et aL obtained from Sigma. All other reagents used were of analytical grade. Purification of Isocitrate Dehydrogenases—IDH-II of Vibrio ABE-1 was purified by means of the same procedures as described previously (10). IDH of V. parahaemolyticus was purified by the procedures described below. Step 1. Crude extract: Frozen cells (about 30 g) were thawed and suspended in 30 ml of 20 mM potassium phosphate buffer (pH 6.8) containing 0.5 M NaCl, 2 mM MgCl2, and 10 mM 2-mercaptoethanol (referred to as buffer A). The cells were disrupted by repeated 1-min bursts of ultrasonic vibration of total 30 min with continuously cooling by ice-water. Crude extract was obtained by centrifugation at 20,000 X g for 20 min. Step 2. Protamine sulfate treatment: Protamine sulfate (2% solution in buffer A) was added dropwise to the crude extract with continuous stirring to give a final concentration of 0.2 mg protamine sulfate/mg protein of the crude extract. After further stirring for 30 min, the mixture was centrifuged at 20,000 X g for 20 min, and the supernatant was saved. Step 3. Ammonium sulfate fractionation: Ammonium sulfate fractionation (0.45 to 0.75 saturation) was performed by adding solid ammonium sulfate to the supernatant after Step 2, as described previously (10). The precipitate formed by adding ammonium sulfate to 0.75 saturation was collected by centrifugation (20,000 X g, 20 min) and dissolved in a small volume of 20 mM potassium phosphate buffer, pH 6.8, containing 2 mM MgCl2, 10 mM 2-mercaptoethanol, 30 mM NaCl, 5 mM tripotassium citrate, and 10% glycerol (referred to as buffer B). The enzyme solution was dialyzed against buffer B with three changes of buffer. Step 4. DEAE-Toyopearl 650M chromatography: The dialyzed enzyme solution was applied to a column of DEAE-Toyopearl 650M (2.6x20 cm) equilibrated with buffer B. The column was washed with 4 column volumes of buffer B, and IDH activity was eluted with a linear gradient of NaCl (30 mM to 200 mM in 750 ml). Fractions containing high activity were combined. The pooled enzyme solution was concentrated with polyethyleneglycol #20,000, and dialyzed against 20 mM potassium phosphate buffer, pH 6.8, containing 2 mM MgCl2, 10 mM 2-mercaptoethanol, 100 mM NaCl, 5 mM tripotassium citrate, and 10% glycerol (referred to as buffer C) with three changes of buffer. Step 5. Phenyl-Sepharose CL-4B chromatography: To the dialyzed enzyme solution was added an equal volume of buffer C containing 2.6 M ammonium sulfate. The enzyme mixture was applied to a column of phenyl-Sepharose CL-4B (2.6 X 12 cm) equilibrated with buffer C containing 1.3 M ammonium sulfate, and then washed with the same buffer. The enzyme activity was eluted by a linearly decreasing gradient of ammonium sulfate concentration from 1.3 to 0.0 M in a total volume of 700 ml. The fractions with high activity were combined, concentrated with polyethyleneglycol #20,000, and dialyzed against 20 mM potassium phosphate buffer, pH 6.8, containing 2 mM MgCl2, l m M 2-mercaptoethanol, 10% glycerol, and 20 mM NaCl (referred to as buffer D) with three changes of buffer. Step 6. Red-Sepharose CL-6B chromatography: The dialyzed enzyme solution was applied to a column of J. Biochem.
851
Isocitrate Dehydrogenase of Vibrio parahaemolyticus Red-Sepharose CL-6B (1X14 cm) equilibrated with buffer D and washed with three column volumes of the same buffer. The column was washed further with buffer D containing 50 mM NaCl instead of 20 mM NaCl until the absorbance at 280 run decreased to almost zero. As shown in Fig. 1, the enzyme activity was eluted with buffer D containing 4 mM trisodium DL-isocitrate instead of 20 mM NaCl. The pooled enzyme solution was concentrated as described above and dialyzed against buffer D without NaCl. All operations described above were performed at 4'C or below. RESULTS Purification of V. parahaemolyticus WH—The temper ature-IDH activity profile of V. parahaemolyticus crude extract showed a single peak at 40-45°C (data not shown). This result suggested that this bacterium does not possess IDH isozymes differing in thermostability, such as IDH-I and -II of Vibrio ABE-1 (20). In addition, we could not detect either NAD+-linked IDH activity in the crude extract or any IDH activity in membrane fraction which was prepared as described {21) (data not shown). In order to investigate the molecular structure and the possible presence of IDH isozymes in V. parahaemolyticus, IDH was purified as described in "MATERIALS AND METHODS."
Throughout the purification, little IDH activity was detected other than that of the main IDH peak. Typical results of the purification are summarized in Table I. The purified IDH was eluted as a single symmetric peak of protein of Mr 80,000 by gel filtration, and showed a single band on native or SDS-polyacrylamide gel electrophoresis (Fig. 2). The migration pattern of the IDH of V. parahaemolyticus on SDS gel was very similar to that of IDH-II (Fig. 2), and the molecular weight of the former was determined as 80,000. This value is the same as that obtained by gel filtration. Analysis of the purified enzyme by isoelectric focusing gave a single band corresponded to a pi value of 5.0. These results indicate that V. parahaemolyticus possesses a single IDH of monomeric form. Amino Acid Composition—The amino acid composition of V. parahaemolyticus IDH is shown in Table II. For comparison, the data for IDH-II (10) are also presented. The content of basic amino acids in V. parahaemolyticus IDH was found to be slightly higher than that of IDH-II and resulted in an acidic/basic amino acid (Glx+Asx/Lys+ Arg) ratio of 1.88, a value which is comparable to those (1.73-1.81) of most prokaryotic IDHs (6).
kDa
Fig. 2. SDS-polyacrylamide gel electrophoresis of V. parahaemolyticus IDH. Protein was electrophoresed under denaturing conditions as described in •MATERIALS AND METHODS.' Lanes 1 and 4, marker proteins; lane 2, pooled fraction (10 ng protein) of Red Sepharose CL-6B; lane 3, purified EDH-n ( 3 ^ g protein) of Vibrio ABE-1. Proteins were stained with Coomassie Brilliant Blue R-250.
72
Elution Volune (ml) Fig. 1. Elution profile of V. parahaemolyticus IDH from a column of Red-Sepharose CL-6B. Details of the procedure were given in "MATERIALS AND METHODS." The start of washing with 50 mM NaCl and that of elution with 4 mM DL-isocitrate are indicated. EDH activity (O) was determined under standard assay conditions as described in "MATERIALS AND METHODS."
TABLE I.
Purification of IDH from V.
Step
Total protein (mg)
Crude extract 5,230 Protamine sulfate 3,090 Ammonium sulfate 1,020 DEAE-Toyopearl 650M 160 Phenyl-Sepharose CL-4B 22.2 Red-Sepharose CL-6B 6.12 Vol. 112, No. 6, 1992
Total activity (units) 5,620 5,470 4,070 4,190 2,530 971
parahaemolyticus. Specific activity Yield (units/mg protein) 1.07 100 1.77 97.3 4.61 72.5 26.2 74.6 114.0 45.0 156.6 17.3
TABLE n . Amino acid composition of V. parahaemolyticus IDH. The results were averaged and the integral numbers of amino acids based on a molecular weight of 80,000 are presented. The data (10) for IDH-n are also presented. V. parahaemolyticus Amino n)H-n acid Integer Mol% Integer Mol% Asx 9.39 70 76 9.99 5.65 Thr 5.29 43 40 9.92 72 9.46 Ser 75 11.37 Glx 83 10.91 86 4.49 4.07 Pro 34 31 8.86 9.99 67 76 Gly 76 10.05 89 11.70 Ala 0.79 5 0.66 Cys 6 7.14 6.04 Val 46 54 1.85 13 Met 14 1.71 4.49 37 4.86 34 lie 8.07 8.02 61 61 Leu 1.59 12 2.50 19 Tyr 3.17 24 3.02 23 Phe 6.48 49 5.91 45 Lys 1.98 15 1.97 15 His 4.49 34 3.02 23 Arg 0.66 4 0.53 Trp 5 Total 761 756
N. Fukunaga et al.
852 Temperature CC)
10
V.p.
Ser Thr Glu Lys Pro Thr l i e l i e Tyr Thr l i e Thr A3p Glu
IDH-II
Ser Thr Asp Asn Ser lys l i e H e Tyr Thr H e Thr Asp Clu
40
30
3.2
3.3
20
10
40
30
3.2
3.3
20
10
Clu Ala Pro Thr l i e Val Trp Thr Arg Thr A3p Git
R.v
15
20
25
V.p.
Ala Pro Ala Leu Ala Thr Tyr SerlAsnlLeu Pro H e IleJArg
IDH-II
Ala Pro Ala Leu Ala Thr Tyr Ser Leu Leu Pro H e H e Glnl
R.v.
Ser|pro Ala Leu Ala|ser|Tyr Ser Leu Leu Pro I l e | v a l | G l n |
Fig. 3. Comparison of N-terminal amino acid sequences of bacterial monomeric IDHs. EDH from V. parahaemolyticus (V.p.), Vibrio ABE-1 (EDH-II), and R vanielii (R.m., 6). Identical residues are boxed.
TABLE m . Immunotltration of IDHs with antl-EDH-I or -II antiserum. The EDH activities of the crude extract were immunotitrated with anti-IDH-I or -II antiserum as described in "MATERIALS AND METHODS." Preimmune rabbit serum was used as the control. Inhibition was expressed as the relative value of the immunotitrated EDH activity to the control. Inhibition (%) IDH structure Bacteria Antiserum (Ref.) IDH-I LDH-n Vibrio ABE-1 EDH-I
0
0.8 0.2 0.8 4.6 1.3 4.3 6.1 4.1
Vibrio parahaemolyticus
0 0
97.2 38.5
Azotobacter vinelandii
2.0
23.4
Escherichia coli Salmonella typhimurium Bacillus subtilis Salmonella paratyphi B Pseudomonas aeruginosa Serratia marcescens Micrococcus lysodeikticus Vibrio ABE-1 EDH-E
100
50.6 53.4 65.4 75.1 75.2 40.8
3.4
3.5
3.6
3.4
3.5
3.6
1/T x 103 CK"1) Fig. 4. Changes of Km values for DL-isocitrate and NADP+ at various temperatures. K^ values of IDH of V, parahaemolyticus (O) and EDH-II of Vibrio ABE-1 (•) were determined at the temperatures indicated.
Dimer (20) Dimer (3) Dimer (27)
Monomer (10) Monomer (This study) Monomer (15) 20
25
30
35
40
Temperature CC) N-Terminal Amino Acid Sequence—The N-terminal sequence up to 28 amino acid residues of V. parahaemolyticus IDH was determined (Fig. 3) and compared to those of IDH-II (20) and Rhodomicrobium vanielii IDH (6), which are the only two other monomeric IDHs to have been partially sequenced to date. There is substantial homology among these three monomeric IDHs: V. parahaemolyticus IDH shows 79% homology with IDH-II and 61% with Rm. v. IDH. Homology with inclusion of conservative substitution in Rm. v. IDH is 71%. On the other hand, the N-terminal region of V. parahaemolyticus IDH showed no homology with the N-terminal region of dimeric IDHs of prokaryotes such as E. coli (13), Thermus thermophUus (22), and Vibrio ABE-1 (20). Immunological Properties—We previously observed (20) that structurally different EDH isozymes (EDH-I and -II) of Vibrio ABE-1 do not share a common antigenicity even though they are closely related to each other in amino acid composition. Thus, EDH of V. parahaemolyticus would be expected to cross-react with the antisera to EDH-II (monomeric enzyme) but not with the antisera to EDH-I (dimeric enzyme). Ouchterlony double immunodiffusion test revealed that a single precipitin line was indeed formed
Fig. 5. Thermostability of V. parahaemolyticus EDH. The purified enzyme (25 ii% protein/ml) was incubated for 20 min in the basal buffer comprising 20 mM potassium phosphate (pH 6.8), 100 mM NaCl, and 2 mM MgCl, supplemented as follows: none (•); 10 mM 2-mercaptoethanol (•); 5 mM potassium citrate (A.); 10% glycerol (•); 10 mM 2-mercaptoethanol, 5 mM potassium citrate, and 10% glycerol (O). After the incubation, the remaining enzyme activity was determined under standard assay conditions as described in •MATERIALS AND METHODS.'
between V. parahaemolyticus EDH and the antisera to EDH-II but not with the anti-EDH-I antisera (data not shown). In the immunotitration test, the antisera to EDH-II caused partial loss of EDH activity in the crude extract of V. parahaemolyticus (Table HI). By kinetic enzyme linked immunoassay, the relative immuno-crossreactivity of the purified EDH of V. parahaemolyticus to the EDH-II was found to be 37%. So, the loss of EDH activity in the crude extract of V. parahaemolyticus might reflect the relative immuno-crossreactivity to EDH-II. As shown in Table HI, EDH activities in the crude extract of several other bacteria than Micrococcus lysodeikticus were specifically immunoneutralized with antisera to either EDH-I or EDH-II. These J. Biochem.
853
Isocitrate Dehydrogenase of Vibrio parahaemolyticus A, nn 210
10
15 20 25 Tine (nin)
30
220
230
240
35
Fig. 6. Time course of heat-denatnration of IDHs. The purified enzymes in 20 mM potassium phosphate, pH 7.0, containing 2 mM MgCli, 100 mM NaCl, and 5 mM potassium citrate, were incubated at the temperatures indicated below. The enzyme concentrations of V. parahaemolyticus and Vibrio ABE-1 were 0.52 and 0.61 mg protein/ ml, respectively. After appropriate time intervals, aliquots of the IDH samples were withdrawn for the determination of the enzyme activity under the standard assay conditions as described in "MATERIALS AND METHODS." V. parahaemolyticus IDH incubated at 30 (•) and 45'C (O), and Vibrio ABE-1 IDH-II at 10 (A) and 30*C ( A ) .
TABLE IV. Contents of helix, £-form, /9-turn, and random form in the native and heat-denatured monomerlc IDHs, computed from the CD spectra shown in Fig. 7, A and B. All values are expressed as percentages. IDH yS-turn Random Helix /J-form Vibrio parahaemolyticus Native 22 25 30 23 Denatured 9 28 49 14 Vibrio ABE-1 IDH-II 17 Native 36 17 31 Denatured 8 30 51 13
results suggest that the examined bacterial strains except Vibrio ABE-1 and M. lysodeikticus may possess either monomeric or dimeric form of IDH. Kinetic Properties—Changes of Km values for both substrate and NADP+ of the purified IDH of V. parahaemolyticus at various temperatures were compared with those of IDH-II. As shown in Fig. 4, the minimum Kn values for NADP+ and substrate were found at 20 and 25 "C, respectively, with V. parahaemolyticus. Since the Arrhenius plot of this enzymatic activity showed a slight change of slope in the temperature range of 20-25'C (data not shown), temperature-induced conformational change might have occurred and influenced the binding of substrate or cofactor to the enzyme in this temperature range. On the other hand, Km values of IDH-II for both coenzyme and substrate increased linearly with elevating temperature. These results indicate a clear difference in the temperature dependency of the enzyme-substrate or cofactor binding exists between the two enzymes. Thermostability—The purified IDH of V. parahaemolyticus showed an optimum temperature for the activity of 40"C in the standard assay system based on Tris/HCl. In the same assay system,- the optimum temperature of IDH-n is 20'C {20). Thermostability of enzymes can be influenced by the presence of chemicals such as salts, Vol. 112, No. 6, 1992
Fig. 7. Circular dichroism spectra of native and heat-denatured IDH of V. parahaemolyticus (A) and DDH-H of Vibrio ABE-1 (B). The composition of the buffer system and concentrations of the enzyme proteins were the same as described in the legend to Fig. 6. The spectra for the native enzymes (O) were measured at 30'C for V. parahaemolyticus and 10'C for Vibrio ABE-1. The spectra for the denatured enzymes (•) were determined at 45'C for V. parahaemolyticus and 30'C for Vibrio ABE-1. Other experimental details were as described in "MATERIALS AND METHODS."
substrates, cofactors, or metal ions. To examine the effects of various chemicals, remaining IDH activity was determined in the standard assay system after the incubation of the enzyme at various temperature in phosphate buffer supplemented with the chemicals (Fig. 5). It was found that the enzyme was stable up to 37"C for at least 20 min when glycerol, 2-mercaptoethanol, and citrate were present all together. Circular Dichroism—To evaluate the characteristics of the secondary structures of V. parahaemolyticus IDH and the IDH-II, we measured the circular dichroism (CD) of both native and denatured forms of the IDHs. As can be seen in Fig. 5, V. parahaemolyticus IDH was almost stable at 30°C in phosphate buffer system supplemented with citrate, but was completely inactivated above 40*C. The thermolabile IDH-U was completely inactivated at 30"C in the same buffer system, but retained its full activity at 10'C. Furthermore, a half of each IDH sample for CD measurement was used to determine the enzyme activity versus time under various temperatures (Fig. 6). Based on these results, the CD was measured at the respective temperatures for the native or denatured state (Fig. 7, A and B). The observed negative minimum of the CD spectra near 220 nm with both native IDHs disappeared when the enzymes were inactivated. Table IV shows the relative contents of secondary structures computed from the CD data. The relative contents of ordered structures in the two native IDHs were similar, though the content of helix was
854
N. Fukunaga et al.
rather less than the values (30-46%) found for the dimeric IDHs of E. coli (23) and cyanobacterium (24). It is interesting to note that the heat-inactivated enzymes still retained a considerable amount of ordered structures (Table IV) and no protein precipitate was formed during the experiments. This result suggests that the heat-inactivation of the enzymes under the experimental conditions described in "MATERIALS AND METHODS" did not result from complete denaturation of the protein structures, but from conformational changes of the active or cofactor binding site of the proteins. When the heat-inactivated IDH samples were shifted to lower temperature (37*C for V.p. IDH or 20'C for IDH-II) and incubated with the addition of isocitrate, about 60% of the original activity was recovered within 1 min. This result indicates that the heat-inactivation process of both IDHs was partially reversible. Previously, we reported similar recovery of the activity from heated and precipitated IDH-II, whereas the heat-inactivation of the dimeric IDH (IDH-I) was irreversible (25). DISCUSSION It is well known that isoforms of enzyme proteins can be separated by using a variety of techniques such as column chromatography, electrophoresis, and differential centrifugation. IDH isozymes (IDH-I and -II) of Vibrio ABE-1 were well separated by column chromatography on DEAESephadex or Butyl-Toyopearl (10). In this study, we could not detect the activity of IDH isozymes differing in thermostability or cofactor specificity in the crude extract of V. parahaemolyticus. In addition, antisera against EDH-I did not cause any loss of IDH activity of the crude extract (Table EEC), and no separable IDH activity peak was found throughout the purification. These results suggest that EDH isozymes are unlikely to exist in V. parahaemolyticus. The purified IDH of V. parahaemolyticus was found to be very similar to the thermolabile monomeric IDH-II of Vibrio ABE-1. Judging from the results of SDS-polyacrylamide gel electrophoresiB (Fig. 2) and gel filtration, IDH of V. parahaemolyticus is a monomeric enzyme. We previously reported that the Vibrio ABE-1 mutant deficient in the activity of the dimeric IDH-I did not show any change in the phenotype of growth-temperature relation or of utilization of carbon sources (12). Taken together, the results suggest that the monomeric IDH may function in the operation of the citric acid cycle in Vibrio ABE-1 and may be distributed widely among other bacterial strains belonging to genus Vibrio. A recent report (6) of the isolation of monomeric IDH from R. vanielii is of great interest, because this bacterium is phylogenically far distant from bacteria of genus Vibrio and its EDH has been shown to have dual coenzyme (NAD+ and NADP+) specificity. As shown in Fig. 3, we confirmed the similarity of the N-terminal sequences up to 28 amino acids of the three different monomeric EDHs. Recently, we have found that the nucleotide sequences of the cloned genes encoding the EDH-I and -II of Vibrio ABE-1 are quite different from each other, though the homology between those of EDH-I and E.coli EDH (23) is very high (unpublished results). This finding may rule out the possibility that the monomeric EDH might have arisen via gene fusion
of the dimeric EDH. Comparison of the available sequence data (6) up to 39 amino acids of the R. vanielii enzyme with the corresponding region of the deduced sequence of EDH-II showed 51% homology between them. Although only a few data are available at the present time, this finding suggests that the monomeric EDHs might have evolved diversely toward enzymes with different thermostability and cofactor specificity without great change in the structural characteristics. Such an evolutionary relationship seems to have also occurred with the group of the dimeric EDHs, because Miyazaki et al. (22) have reported the existence of significant similarity between the complete sequences of E. coli EDH and Thermus thermophilus EDH, which is an extremely thermostable dimeric enzyme with dual coenzyme specificity. The results of immunotitration test with antisera to EDH-I and -H of Vibrio ABE-1 (Table HI) clearly indicate that the immunoantigenicities are well conserved with the two groups of monomeric and dimeric IDHs. The structural properties of the Bacillus subtilis and Salmonella paratyphi EDIIs are not available yet, but the IDHs of the phylogenically related B. stearothermophilus and S. typhimurium have been reported to be dimers (26, 27). Judging from the immuno-crossreactivity, the EDHs of Pseudomonas aeruginosa and Serratia marcescens may be dimers. It is not known at present why the enzyme of Micrococcus lysodeikticus did not crossreact with the antisera of the EDH isozymes of Vibrio ABE-1. The E. coli enzyme is the only EDH which has been crystallized and analyzed in detail (23). From a comparative study of the sequences of the cloned genes encoding T. thermophilus and E. coli EDHs, Miyazaki et aL (22) have suggested that the tertiary structures of the two enzymes are similar. We also observed that the V. parahaemolyticus EDH and EDH-II have similar characteristics in secondary structure (Fig. 7, A and B). Thus, the structural similarity within each of the monomeric and dimeric EDHs seems to extend to the higher structure. The above results indicate a clear distinction between the monomeric and dimeric enzymes, and similarity within each group. We would like to emphasize the uniqueness of Vibrio ABE-1 in possessing both EDH isozymes, although the meaning of this coexistence is unknown. Much further study will be required for elucidation of the relation between the two distinct groups of enzymes. For comparison with the well studied dimeric EDH of E. coli, we are attempting to obtain a good crystal of the monomeric enzyme, and an analysis of cloned genes of the EDH isozymes of Vibrio ABE-1 is also in progress in our laboratory. The authors thank Dr. Kazuyuki Kimura, Department of Microbiology, Faculty of Medicine, Hokkaido University, for his kind gift of Vibrio parahaemolyticus Y-4. The authors also thank Dr. Katsutosi Nitta, Department of Polymer, Faculty of Science, Hokkaido University for his advice on circular dichroism measurement.
REFERENCES 1. Colman, R.F. (1983) PepL Protein Rev. 1, 41-69 2. Howard, RX. & Becker, R.R. (1970) J. BioL Chem. 246, 31863194 3. Burke, W.F., Johanson, R.A., & Reeves, H.C. (1974) Biochim. Biophys. Ada 353, 22-36 4. Buzdygon, B.E., Braginski, J.E., & Chung, A.E. (1973) Arch. J. Biochan.
Isocitrate Dehydrogenase of Vibrio parahaemolyticus Biochem. Biophys. 159, 400-408 5. Eguchi, H., Wakagi, T., & Oshima, T. (1989) Biochim. Biophys. Ada 990, 133-137 6. Leyland, MX. & Kelly, D. (1991) Eur. J. Biochem. 202, 85-93 7. Hampton, M.L. & Hanson, R.S. (1969) Biochem. Biophys. Res. Common. 36, 296-305 8. Greenfield, S. & Claus, G.W. (1969) J. Bacteriol. 100, 12641270 9. Self, C.H., Weitzman, P.D.J., & Parker, M.G. (1973) Biochem. J. 132, 215-221 10. Ifihii, A., Imagawa, S., Fukunaga, N., Sasaki, S., Minowa, O., Mizuno, Y., & Shiokawa, H. (1987) J. Biochem. 102,1489- 1498 11. Takada, Y., Ochiai, T., Okuyama, H., Nishi, K., & Sasaki, S. (1979) J. Gen. Appl. Microbiol. 26, 11-19 12. Fukunaga, N., Yoshida, S., Ishii, A., Imagawa, S., Takada, Y., & Sasaki, S. (1988) J. Gen. Appl. Microbiol. 34, 457-465 13. Thorsness, P.E. & Koahland, D.E., Jr. (1987) J. Biol Chem. 262, 10422-10425 14. Cupp, J.R. & McAlister-Henn, L. (1991) J. Biol. Chem. 286, 22199-22205 15. Chung, A.E. & Franzen, J.S. (1969) Biochemistry 8, 3175-3184 16. Bradford, M.M. (1976) Anal. Biochem. 72, 248-254 17. Tsnag, V.C.W., Wilson, B.C., & Peralta, J.M. (1983) Methods
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855 Enzymol. 92, 391-403 18. Yang, J.T., Wu, C.-S.C, & Martines, H.M. (1986) Methods Enzymol. 130, 208-269 19. Chung, C.T., Wu, C.-S.C, & Yang, J.T. (1978) Anal Biochem. 91, 13-31 20. Ochiai, T., Fukunaga, N., & Sasaki, S. (1979) J. Biochem. 86, 377-384 21. Wada, M., Fukunaga, N., & Sasaki, S. (1989) J. Bacteriol. 171, 4267-4271 22. Miyazaki, K., Eguchi, H., Yamagishi, A., Wakagi, T., & Oahima, T. (1992) AppL Environ. Microbiol. 58, 93-98 23. Hurley, J.H., Thorsness, P.E., Ramalingam, V., Helmers, N.H., Koshland, D.E., Jr., & Stroud, R.M. (1989) Proc. Natl. Acad. Sci. USA 86, 8635-8639 24. Muro-Pastor, M.I. & Florencio, F. J. (1992) Eur. J. Biochem. 203, 99-105 25. Ochiai, T., Fukunaga, N., & Sasaki, S. (1984) J. Gen. Appl. Microbiol. 30, 479-487 26. Nagaoka, T., Hachimori, A., Takeda, A., & Samejima, T. (1977) J. Biochem. 81, 71-78 27. Wang, J.Y.J. & Koshland, D.E., Jr. (1972) Arch. Biochem. Biophys. 363, 357-367
Purification and Characterization of Monomeric Isocitrate Dehydrogenase with NADP+-Specificity from Vibrio parahaemolyticus Y-4 Noriyuki Fukunaga,1 Sigeki Imagawa,2 Takehiko Sahara, Atsushi Ishii, 3 and Masahiro Suzuki Department of Biology, Faculty of Science, Hokkaido University, Kita-ku, Sapporo, Hokkaido 060 Received for publication, April 15, 1992
NADP+-dependent isocitrate dehydrogenase [IDH: EC 1.1.1.42] was purified to electrophoretic homogeneity from Vibrio parahaemolyticus Y-4, and shown to be a monomeric protein of molecular weight 80,000 with a pi of 5.0. The amino acid composition and partial sequence at the N-terminus resembled those reported for other bacterial monomeric IDHs. Immunotitration with antisera to the monomeric and dimeric enzymes (antisera to EDH-II and -I of Vibrio ABE-1) showed an iTnTniinof.hftmip.nl distinction between the monomeric and dimeric IDHs, but there is similarity within the IDHs of each group. The circular dichroism spectra of the native and heat-denatured enzyme are also similar to those of monomeric IDH (IDH-II of Vibrio ABE-1). These monomeric IDHs are proteins comprising 17-22% helix and 25-35% £-pleated sheet in the native state.
Isocitrate dehydrogenase (IDH) catalyzes the oxidative decarboxylation of isocitrate to 2-oxoglutarate and C0 2 , and is distributed in a wide variety of organisms. In eukaryotic cells, more than two isozymes of IDH differing in their intracellular localizations, structures, and coenzyme specificities are present (2). However, bacterial IDHs from many sources are NADP+-specific (e.g. 2-4) or show much higher affinity to NADP+ than to NAD+ as a cofactor (5, 6), although NAD+-linked IDH is present in a few bacteria (7, 8). In contrast to eukaryotic cells, most bacteria were widely believed to possess either monomeric or dimeric form of IDH, until structurally different IDH isozymes were reported in Acinetobacter calcoaceticus (9) and a psychrophilic marine bacterium, Vibrio sp. strain ABE-1 (Vibrio ABE-1, 10), isolated from sea water off the coast of Hokkaido (11). We previously reported (10) that NADF^-specific IDH-I and -II of Vibrio ABE-1 are a thermostable dimeric form consisting of homologous subunits of MT 46,000 and a thermolabile monomeric form of Mr 80,000, respectively. Furthermore, the N-terminal amino acid sequences of the EDH isozymes (10, 12) are quite different from each other, though that of the IDH-I is similar to that of Escfierichia coli IDH, which is a dimer (23). To our knowledge, coexistence of such different IDH isozymes has not been reported in any other bacterial strain. Recently, Leyland and Kelly (6) reported that the IDH of the photosynthetic bacterium, Rhodomicrvbium vanielii, is a monomer and its N-terminal amino acid sequence is similar to that of the IDH-II. Based on the comparison between amino acid sequences of E. coli EDH and yeast mitochondria! IDH, which is an NAD+-specific octamer consisting of two non-identical subunits, Cupp and Mc1 To whom correspondence should be addressed. Present addresses: * Niigata Research Laboratory, Mitsubishi Gas Co., Niigata 950-31; "Group on Neuropharmacology, Faculty of Pharmacy, Josai University, Sakado, Saitama 350-02.
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Alister-Henn (14) indicated that one of the subunits of the yeast IDH contains a region similar in sequence to that of the E. coli IDH. These results have implications for the molecular evolution of IDH. The goal of our study is to elucidate the physiological and evolutional meaning of the coexistence of the IDH isozymes in Vibrio ABE-1. Initially, we investigated whether another bacterial strain belonging to genus Vibrio possesses structurally different IDH isozymes like those of Vibrio ABE-1. For this purpose, we purified EDH from Vibrio parahaemolyticus Y-4, because this bacterium is a mesophile with slightly halophilic nature. We found that this bacterium possesses a single EDH of monomeric form with similar N-terminal amino acid sequence, immuno-crossreactivity, and secondary structure to those of EDH-II. MATERIALS AND METHODS Bacteria and Culture Conditions—Vibrio ABE-1 (22) and V. parahaemolyticus Y-4 (V. parahaemolyticus, given to us by Dr. Kazuyuki Kimura of the Department of Microbiology, Faculty of Medicine, Hokkaido University) were cultured with vigorous shaking in a nutrient medium consisting of 1% peptone and meat extract with 0.5 M NaCl at 15 and 37"C, respectively. Azotobacter uinelandii strain LAM 1078 (kindly donated by the Institute of Applied Microbiology, The University of Tokyo) was cultured as described (15). The other bacterial strains used in this study were cultured at the respective optimum temperatures in the nutrient media described above, but 50 mM NaCl was used. Cells in the early stationary phase were harvested and washed three times with chilled 0.5 M NaCl, then frozen at — 80*C until use. Assay of IDH Activity—NADP+-EDH activity was determined spectrophotometrically at 20'C for EDH-EI or at 37°C for V. parahaemolyticus EDH unless otherwise stated. Components of the reaction mixture were the same as
850 described previously (20). The assay system for NAD+IDH was the same as the NADP+-IDH assay system except that NAD+ was used instead of NADP+ and at least 10 times more enzyme protein was employed. One unit of the enzyme activity is defined as the amount of enzyme catalyzing the formation of 1 /*mol of product per min. Protein concentrations were determined by the method of Bradford (16) with bovine y-globulin as a standard. Electrophoresis—Conditions for native and denaturing polyacrylamide gel electrophoresis were the same as described previously (20). Proteins were stained with Coomassie Brilliant Blue R-250. Analytical isoelectric focusing was performed with a slab gel of 5% acrylamide containing 2% Ampholine of pH 3.5-10, and 4 mM isocitrate as described previously (10). Amino Acid Analysis and N-Terminal Sequencing— Purified IDH protein was hydrolyzed in 6 M HC1 at 110'C for 24, 48, and 72 h. The hydrolysates were analyzed with a Hitachi model 835 amino acid analyzer. Cysteine, methionine, and tryptophan were estimated as described previously (10). Amino acid sequencing was performed by automated Edman degradation with an Applied Biosystems 477A gas phase protein sequencer. Immunological Studies—Rabbit antisera and IgG fractions against the IDH-I and -II were obtained by the procedure described previously (10), and frozen at —80'C until use. Immunotitration with each of the above antisera was performed by essentially the same method as described previously (10). For the buffer system, 20 mM potassium phosphate (pH 6.5) containing 0.5 M NaCl, 2 mM MgCl2, 1 mM EDTA, and 10 mM 2-mercaptoethanol was used in the experiments. IDH activity of the crude extract was adjusted to 0.3-0.5 unit/ml, and the antiIDH-I and -II antisera were diluted to 2- and 4-fold, respectively, with the above buffer containing 1% bovine serum albumin. The relative immunocross-reactivity between the purified IDH of V. parahaemolyticus and IDH-II of Vibrio ABE-1 was determined by the method of quantitative, single tube, kinetic-dependent enzyme-linked immunosorbent assay as described (17). Measurement of Circular Dichroism—Circular dichroism (CD) was measured with a Jasco J-20 spectropolarimeter at desired temperatures under constant nitrogen flush. IDH samples (about 0.5 mg protein/ml of 20 mM potassium phosphate buffer, pH 7.0, containing 100 mM NaCl, 5 mM tripotassium citrate, and 2 mM MgCl2) were divided into two portions. Half of each sample was used for CD measurement, and the other half to check the enzyme activity. Prior to the CD measurement, the IDH samples were incubated at the desired temperature for 30 min to attain temperature equilibration. The data were expressed in terms of mean residue eUiptdcity [0]. In order to* estimate secondary structure, we employed a CD spectrum analysis program (28) based on the Chung-Wu-Yang method (29). Chemicals—DEAE-Toyopearl 650M was purchased from Tosoh. Phenyl-Sepharose CL-6B, Red-Sepharose CL-6B, and pi marker proteins were obtained from Pharmacia. Marker proteins for molecular weight were purchased from Boehringer-Mannheim. NAD+ and NADP+ were the products of Oriental Yeast. Ampholine, o-dianisidine, and goat anti-rabbit-IgG-peroxidase conjugate were
N. Fukunaga et aL obtained from Sigma. All other reagents used were of analytical grade. Purification of Isocitrate Dehydrogenases—IDH-II of Vibrio ABE-1 was purified by means of the same procedures as described previously (10). IDH of V. parahaemolyticus was purified by the procedures described below. Step 1. Crude extract: Frozen cells (about 30 g) were thawed and suspended in 30 ml of 20 mM potassium phosphate buffer (pH 6.8) containing 0.5 M NaCl, 2 mM MgCl2, and 10 mM 2-mercaptoethanol (referred to as buffer A). The cells were disrupted by repeated 1-min bursts of ultrasonic vibration of total 30 min with continuously cooling by ice-water. Crude extract was obtained by centrifugation at 20,000 X g for 20 min. Step 2. Protamine sulfate treatment: Protamine sulfate (2% solution in buffer A) was added dropwise to the crude extract with continuous stirring to give a final concentration of 0.2 mg protamine sulfate/mg protein of the crude extract. After further stirring for 30 min, the mixture was centrifuged at 20,000 X g for 20 min, and the supernatant was saved. Step 3. Ammonium sulfate fractionation: Ammonium sulfate fractionation (0.45 to 0.75 saturation) was performed by adding solid ammonium sulfate to the supernatant after Step 2, as described previously (10). The precipitate formed by adding ammonium sulfate to 0.75 saturation was collected by centrifugation (20,000 X g, 20 min) and dissolved in a small volume of 20 mM potassium phosphate buffer, pH 6.8, containing 2 mM MgCl2, 10 mM 2-mercaptoethanol, 30 mM NaCl, 5 mM tripotassium citrate, and 10% glycerol (referred to as buffer B). The enzyme solution was dialyzed against buffer B with three changes of buffer. Step 4. DEAE-Toyopearl 650M chromatography: The dialyzed enzyme solution was applied to a column of DEAE-Toyopearl 650M (2.6x20 cm) equilibrated with buffer B. The column was washed with 4 column volumes of buffer B, and IDH activity was eluted with a linear gradient of NaCl (30 mM to 200 mM in 750 ml). Fractions containing high activity were combined. The pooled enzyme solution was concentrated with polyethyleneglycol #20,000, and dialyzed against 20 mM potassium phosphate buffer, pH 6.8, containing 2 mM MgCl2, 10 mM 2-mercaptoethanol, 100 mM NaCl, 5 mM tripotassium citrate, and 10% glycerol (referred to as buffer C) with three changes of buffer. Step 5. Phenyl-Sepharose CL-4B chromatography: To the dialyzed enzyme solution was added an equal volume of buffer C containing 2.6 M ammonium sulfate. The enzyme mixture was applied to a column of phenyl-Sepharose CL-4B (2.6 X 12 cm) equilibrated with buffer C containing 1.3 M ammonium sulfate, and then washed with the same buffer. The enzyme activity was eluted by a linearly decreasing gradient of ammonium sulfate concentration from 1.3 to 0.0 M in a total volume of 700 ml. The fractions with high activity were combined, concentrated with polyethyleneglycol #20,000, and dialyzed against 20 mM potassium phosphate buffer, pH 6.8, containing 2 mM MgCl2, l m M 2-mercaptoethanol, 10% glycerol, and 20 mM NaCl (referred to as buffer D) with three changes of buffer. Step 6. Red-Sepharose CL-6B chromatography: The dialyzed enzyme solution was applied to a column of J. Biochem.
851
Isocitrate Dehydrogenase of Vibrio parahaemolyticus Red-Sepharose CL-6B (1X14 cm) equilibrated with buffer D and washed with three column volumes of the same buffer. The column was washed further with buffer D containing 50 mM NaCl instead of 20 mM NaCl until the absorbance at 280 run decreased to almost zero. As shown in Fig. 1, the enzyme activity was eluted with buffer D containing 4 mM trisodium DL-isocitrate instead of 20 mM NaCl. The pooled enzyme solution was concentrated as described above and dialyzed against buffer D without NaCl. All operations described above were performed at 4'C or below. RESULTS Purification of V. parahaemolyticus WH—The temper ature-IDH activity profile of V. parahaemolyticus crude extract showed a single peak at 40-45°C (data not shown). This result suggested that this bacterium does not possess IDH isozymes differing in thermostability, such as IDH-I and -II of Vibrio ABE-1 (20). In addition, we could not detect either NAD+-linked IDH activity in the crude extract or any IDH activity in membrane fraction which was prepared as described {21) (data not shown). In order to investigate the molecular structure and the possible presence of IDH isozymes in V. parahaemolyticus, IDH was purified as described in "MATERIALS AND METHODS."
Throughout the purification, little IDH activity was detected other than that of the main IDH peak. Typical results of the purification are summarized in Table I. The purified IDH was eluted as a single symmetric peak of protein of Mr 80,000 by gel filtration, and showed a single band on native or SDS-polyacrylamide gel electrophoresis (Fig. 2). The migration pattern of the IDH of V. parahaemolyticus on SDS gel was very similar to that of IDH-II (Fig. 2), and the molecular weight of the former was determined as 80,000. This value is the same as that obtained by gel filtration. Analysis of the purified enzyme by isoelectric focusing gave a single band corresponded to a pi value of 5.0. These results indicate that V. parahaemolyticus possesses a single IDH of monomeric form. Amino Acid Composition—The amino acid composition of V. parahaemolyticus IDH is shown in Table II. For comparison, the data for IDH-II (10) are also presented. The content of basic amino acids in V. parahaemolyticus IDH was found to be slightly higher than that of IDH-II and resulted in an acidic/basic amino acid (Glx+Asx/Lys+ Arg) ratio of 1.88, a value which is comparable to those (1.73-1.81) of most prokaryotic IDHs (6).
kDa
Fig. 2. SDS-polyacrylamide gel electrophoresis of V. parahaemolyticus IDH. Protein was electrophoresed under denaturing conditions as described in •MATERIALS AND METHODS.' Lanes 1 and 4, marker proteins; lane 2, pooled fraction (10 ng protein) of Red Sepharose CL-6B; lane 3, purified EDH-n ( 3 ^ g protein) of Vibrio ABE-1. Proteins were stained with Coomassie Brilliant Blue R-250.
72
Elution Volune (ml) Fig. 1. Elution profile of V. parahaemolyticus IDH from a column of Red-Sepharose CL-6B. Details of the procedure were given in "MATERIALS AND METHODS." The start of washing with 50 mM NaCl and that of elution with 4 mM DL-isocitrate are indicated. EDH activity (O) was determined under standard assay conditions as described in "MATERIALS AND METHODS."
TABLE I.
Purification of IDH from V.
Step
Total protein (mg)
Crude extract 5,230 Protamine sulfate 3,090 Ammonium sulfate 1,020 DEAE-Toyopearl 650M 160 Phenyl-Sepharose CL-4B 22.2 Red-Sepharose CL-6B 6.12 Vol. 112, No. 6, 1992
Total activity (units) 5,620 5,470 4,070 4,190 2,530 971
parahaemolyticus. Specific activity Yield (units/mg protein) 1.07 100 1.77 97.3 4.61 72.5 26.2 74.6 114.0 45.0 156.6 17.3
TABLE n . Amino acid composition of V. parahaemolyticus IDH. The results were averaged and the integral numbers of amino acids based on a molecular weight of 80,000 are presented. The data (10) for IDH-n are also presented. V. parahaemolyticus Amino n)H-n acid Integer Mol% Integer Mol% Asx 9.39 70 76 9.99 5.65 Thr 5.29 43 40 9.92 72 9.46 Ser 75 11.37 Glx 83 10.91 86 4.49 4.07 Pro 34 31 8.86 9.99 67 76 Gly 76 10.05 89 11.70 Ala 0.79 5 0.66 Cys 6 7.14 6.04 Val 46 54 1.85 13 Met 14 1.71 4.49 37 4.86 34 lie 8.07 8.02 61 61 Leu 1.59 12 2.50 19 Tyr 3.17 24 3.02 23 Phe 6.48 49 5.91 45 Lys 1.98 15 1.97 15 His 4.49 34 3.02 23 Arg 0.66 4 0.53 Trp 5 Total 761 756
N. Fukunaga et al.
852 Temperature CC)
10
V.p.
Ser Thr Glu Lys Pro Thr l i e l i e Tyr Thr l i e Thr A3p Glu
IDH-II
Ser Thr Asp Asn Ser lys l i e H e Tyr Thr H e Thr Asp Clu
40
30
3.2
3.3
20
10
40
30
3.2
3.3
20
10
Clu Ala Pro Thr l i e Val Trp Thr Arg Thr A3p Git
R.v
15
20
25
V.p.
Ala Pro Ala Leu Ala Thr Tyr SerlAsnlLeu Pro H e IleJArg
IDH-II
Ala Pro Ala Leu Ala Thr Tyr Ser Leu Leu Pro H e H e Glnl
R.v.
Ser|pro Ala Leu Ala|ser|Tyr Ser Leu Leu Pro I l e | v a l | G l n |
Fig. 3. Comparison of N-terminal amino acid sequences of bacterial monomeric IDHs. EDH from V. parahaemolyticus (V.p.), Vibrio ABE-1 (EDH-II), and R vanielii (R.m., 6). Identical residues are boxed.
TABLE m . Immunotltration of IDHs with antl-EDH-I or -II antiserum. The EDH activities of the crude extract were immunotitrated with anti-IDH-I or -II antiserum as described in "MATERIALS AND METHODS." Preimmune rabbit serum was used as the control. Inhibition was expressed as the relative value of the immunotitrated EDH activity to the control. Inhibition (%) IDH structure Bacteria Antiserum (Ref.) IDH-I LDH-n Vibrio ABE-1 EDH-I
0
0.8 0.2 0.8 4.6 1.3 4.3 6.1 4.1
Vibrio parahaemolyticus
0 0
97.2 38.5
Azotobacter vinelandii
2.0
23.4
Escherichia coli Salmonella typhimurium Bacillus subtilis Salmonella paratyphi B Pseudomonas aeruginosa Serratia marcescens Micrococcus lysodeikticus Vibrio ABE-1 EDH-E
100
50.6 53.4 65.4 75.1 75.2 40.8
3.4
3.5
3.6
3.4
3.5
3.6
1/T x 103 CK"1) Fig. 4. Changes of Km values for DL-isocitrate and NADP+ at various temperatures. K^ values of IDH of V, parahaemolyticus (O) and EDH-II of Vibrio ABE-1 (•) were determined at the temperatures indicated.
Dimer (20) Dimer (3) Dimer (27)
Monomer (10) Monomer (This study) Monomer (15) 20
25
30
35
40
Temperature CC) N-Terminal Amino Acid Sequence—The N-terminal sequence up to 28 amino acid residues of V. parahaemolyticus IDH was determined (Fig. 3) and compared to those of IDH-II (20) and Rhodomicrobium vanielii IDH (6), which are the only two other monomeric IDHs to have been partially sequenced to date. There is substantial homology among these three monomeric IDHs: V. parahaemolyticus IDH shows 79% homology with IDH-II and 61% with Rm. v. IDH. Homology with inclusion of conservative substitution in Rm. v. IDH is 71%. On the other hand, the N-terminal region of V. parahaemolyticus IDH showed no homology with the N-terminal region of dimeric IDHs of prokaryotes such as E. coli (13), Thermus thermophUus (22), and Vibrio ABE-1 (20). Immunological Properties—We previously observed (20) that structurally different EDH isozymes (EDH-I and -II) of Vibrio ABE-1 do not share a common antigenicity even though they are closely related to each other in amino acid composition. Thus, EDH of V. parahaemolyticus would be expected to cross-react with the antisera to EDH-II (monomeric enzyme) but not with the antisera to EDH-I (dimeric enzyme). Ouchterlony double immunodiffusion test revealed that a single precipitin line was indeed formed
Fig. 5. Thermostability of V. parahaemolyticus EDH. The purified enzyme (25 ii% protein/ml) was incubated for 20 min in the basal buffer comprising 20 mM potassium phosphate (pH 6.8), 100 mM NaCl, and 2 mM MgCl, supplemented as follows: none (•); 10 mM 2-mercaptoethanol (•); 5 mM potassium citrate (A.); 10% glycerol (•); 10 mM 2-mercaptoethanol, 5 mM potassium citrate, and 10% glycerol (O). After the incubation, the remaining enzyme activity was determined under standard assay conditions as described in •MATERIALS AND METHODS.'
between V. parahaemolyticus EDH and the antisera to EDH-II but not with the anti-EDH-I antisera (data not shown). In the immunotitration test, the antisera to EDH-II caused partial loss of EDH activity in the crude extract of V. parahaemolyticus (Table HI). By kinetic enzyme linked immunoassay, the relative immuno-crossreactivity of the purified EDH of V. parahaemolyticus to the EDH-II was found to be 37%. So, the loss of EDH activity in the crude extract of V. parahaemolyticus might reflect the relative immuno-crossreactivity to EDH-II. As shown in Table HI, EDH activities in the crude extract of several other bacteria than Micrococcus lysodeikticus were specifically immunoneutralized with antisera to either EDH-I or EDH-II. These J. Biochem.
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Isocitrate Dehydrogenase of Vibrio parahaemolyticus A, nn 210
10
15 20 25 Tine (nin)
30
220
230
240
35
Fig. 6. Time course of heat-denatnration of IDHs. The purified enzymes in 20 mM potassium phosphate, pH 7.0, containing 2 mM MgCli, 100 mM NaCl, and 5 mM potassium citrate, were incubated at the temperatures indicated below. The enzyme concentrations of V. parahaemolyticus and Vibrio ABE-1 were 0.52 and 0.61 mg protein/ ml, respectively. After appropriate time intervals, aliquots of the IDH samples were withdrawn for the determination of the enzyme activity under the standard assay conditions as described in "MATERIALS AND METHODS." V. parahaemolyticus IDH incubated at 30 (•) and 45'C (O), and Vibrio ABE-1 IDH-II at 10 (A) and 30*C ( A ) .
TABLE IV. Contents of helix, £-form, /9-turn, and random form in the native and heat-denatured monomerlc IDHs, computed from the CD spectra shown in Fig. 7, A and B. All values are expressed as percentages. IDH yS-turn Random Helix /J-form Vibrio parahaemolyticus Native 22 25 30 23 Denatured 9 28 49 14 Vibrio ABE-1 IDH-II 17 Native 36 17 31 Denatured 8 30 51 13
results suggest that the examined bacterial strains except Vibrio ABE-1 and M. lysodeikticus may possess either monomeric or dimeric form of IDH. Kinetic Properties—Changes of Km values for both substrate and NADP+ of the purified IDH of V. parahaemolyticus at various temperatures were compared with those of IDH-II. As shown in Fig. 4, the minimum Kn values for NADP+ and substrate were found at 20 and 25 "C, respectively, with V. parahaemolyticus. Since the Arrhenius plot of this enzymatic activity showed a slight change of slope in the temperature range of 20-25'C (data not shown), temperature-induced conformational change might have occurred and influenced the binding of substrate or cofactor to the enzyme in this temperature range. On the other hand, Km values of IDH-II for both coenzyme and substrate increased linearly with elevating temperature. These results indicate a clear difference in the temperature dependency of the enzyme-substrate or cofactor binding exists between the two enzymes. Thermostability—The purified IDH of V. parahaemolyticus showed an optimum temperature for the activity of 40"C in the standard assay system based on Tris/HCl. In the same assay system,- the optimum temperature of IDH-n is 20'C {20). Thermostability of enzymes can be influenced by the presence of chemicals such as salts, Vol. 112, No. 6, 1992
Fig. 7. Circular dichroism spectra of native and heat-denatured IDH of V. parahaemolyticus (A) and DDH-H of Vibrio ABE-1 (B). The composition of the buffer system and concentrations of the enzyme proteins were the same as described in the legend to Fig. 6. The spectra for the native enzymes (O) were measured at 30'C for V. parahaemolyticus and 10'C for Vibrio ABE-1. The spectra for the denatured enzymes (•) were determined at 45'C for V. parahaemolyticus and 30'C for Vibrio ABE-1. Other experimental details were as described in "MATERIALS AND METHODS."
substrates, cofactors, or metal ions. To examine the effects of various chemicals, remaining IDH activity was determined in the standard assay system after the incubation of the enzyme at various temperature in phosphate buffer supplemented with the chemicals (Fig. 5). It was found that the enzyme was stable up to 37"C for at least 20 min when glycerol, 2-mercaptoethanol, and citrate were present all together. Circular Dichroism—To evaluate the characteristics of the secondary structures of V. parahaemolyticus IDH and the IDH-II, we measured the circular dichroism (CD) of both native and denatured forms of the IDHs. As can be seen in Fig. 5, V. parahaemolyticus IDH was almost stable at 30°C in phosphate buffer system supplemented with citrate, but was completely inactivated above 40*C. The thermolabile IDH-U was completely inactivated at 30"C in the same buffer system, but retained its full activity at 10'C. Furthermore, a half of each IDH sample for CD measurement was used to determine the enzyme activity versus time under various temperatures (Fig. 6). Based on these results, the CD was measured at the respective temperatures for the native or denatured state (Fig. 7, A and B). The observed negative minimum of the CD spectra near 220 nm with both native IDHs disappeared when the enzymes were inactivated. Table IV shows the relative contents of secondary structures computed from the CD data. The relative contents of ordered structures in the two native IDHs were similar, though the content of helix was
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rather less than the values (30-46%) found for the dimeric IDHs of E. coli (23) and cyanobacterium (24). It is interesting to note that the heat-inactivated enzymes still retained a considerable amount of ordered structures (Table IV) and no protein precipitate was formed during the experiments. This result suggests that the heat-inactivation of the enzymes under the experimental conditions described in "MATERIALS AND METHODS" did not result from complete denaturation of the protein structures, but from conformational changes of the active or cofactor binding site of the proteins. When the heat-inactivated IDH samples were shifted to lower temperature (37*C for V.p. IDH or 20'C for IDH-II) and incubated with the addition of isocitrate, about 60% of the original activity was recovered within 1 min. This result indicates that the heat-inactivation process of both IDHs was partially reversible. Previously, we reported similar recovery of the activity from heated and precipitated IDH-II, whereas the heat-inactivation of the dimeric IDH (IDH-I) was irreversible (25). DISCUSSION It is well known that isoforms of enzyme proteins can be separated by using a variety of techniques such as column chromatography, electrophoresis, and differential centrifugation. IDH isozymes (IDH-I and -II) of Vibrio ABE-1 were well separated by column chromatography on DEAESephadex or Butyl-Toyopearl (10). In this study, we could not detect the activity of IDH isozymes differing in thermostability or cofactor specificity in the crude extract of V. parahaemolyticus. In addition, antisera against EDH-I did not cause any loss of IDH activity of the crude extract (Table EEC), and no separable IDH activity peak was found throughout the purification. These results suggest that EDH isozymes are unlikely to exist in V. parahaemolyticus. The purified IDH of V. parahaemolyticus was found to be very similar to the thermolabile monomeric IDH-II of Vibrio ABE-1. Judging from the results of SDS-polyacrylamide gel electrophoresiB (Fig. 2) and gel filtration, IDH of V. parahaemolyticus is a monomeric enzyme. We previously reported that the Vibrio ABE-1 mutant deficient in the activity of the dimeric IDH-I did not show any change in the phenotype of growth-temperature relation or of utilization of carbon sources (12). Taken together, the results suggest that the monomeric IDH may function in the operation of the citric acid cycle in Vibrio ABE-1 and may be distributed widely among other bacterial strains belonging to genus Vibrio. A recent report (6) of the isolation of monomeric IDH from R. vanielii is of great interest, because this bacterium is phylogenically far distant from bacteria of genus Vibrio and its EDH has been shown to have dual coenzyme (NAD+ and NADP+) specificity. As shown in Fig. 3, we confirmed the similarity of the N-terminal sequences up to 28 amino acids of the three different monomeric EDHs. Recently, we have found that the nucleotide sequences of the cloned genes encoding the EDH-I and -II of Vibrio ABE-1 are quite different from each other, though the homology between those of EDH-I and E.coli EDH (23) is very high (unpublished results). This finding may rule out the possibility that the monomeric EDH might have arisen via gene fusion
of the dimeric EDH. Comparison of the available sequence data (6) up to 39 amino acids of the R. vanielii enzyme with the corresponding region of the deduced sequence of EDH-II showed 51% homology between them. Although only a few data are available at the present time, this finding suggests that the monomeric EDHs might have evolved diversely toward enzymes with different thermostability and cofactor specificity without great change in the structural characteristics. Such an evolutionary relationship seems to have also occurred with the group of the dimeric EDHs, because Miyazaki et al. (22) have reported the existence of significant similarity between the complete sequences of E. coli EDH and Thermus thermophilus EDH, which is an extremely thermostable dimeric enzyme with dual coenzyme specificity. The results of immunotitration test with antisera to EDH-I and -H of Vibrio ABE-1 (Table HI) clearly indicate that the immunoantigenicities are well conserved with the two groups of monomeric and dimeric IDHs. The structural properties of the Bacillus subtilis and Salmonella paratyphi EDIIs are not available yet, but the IDHs of the phylogenically related B. stearothermophilus and S. typhimurium have been reported to be dimers (26, 27). Judging from the immuno-crossreactivity, the EDHs of Pseudomonas aeruginosa and Serratia marcescens may be dimers. It is not known at present why the enzyme of Micrococcus lysodeikticus did not crossreact with the antisera of the EDH isozymes of Vibrio ABE-1. The E. coli enzyme is the only EDH which has been crystallized and analyzed in detail (23). From a comparative study of the sequences of the cloned genes encoding T. thermophilus and E. coli EDHs, Miyazaki et aL (22) have suggested that the tertiary structures of the two enzymes are similar. We also observed that the V. parahaemolyticus EDH and EDH-II have similar characteristics in secondary structure (Fig. 7, A and B). Thus, the structural similarity within each of the monomeric and dimeric EDHs seems to extend to the higher structure. The above results indicate a clear distinction between the monomeric and dimeric enzymes, and similarity within each group. We would like to emphasize the uniqueness of Vibrio ABE-1 in possessing both EDH isozymes, although the meaning of this coexistence is unknown. Much further study will be required for elucidation of the relation between the two distinct groups of enzymes. For comparison with the well studied dimeric EDH of E. coli, we are attempting to obtain a good crystal of the monomeric enzyme, and an analysis of cloned genes of the EDH isozymes of Vibrio ABE-1 is also in progress in our laboratory. The authors thank Dr. Kazuyuki Kimura, Department of Microbiology, Faculty of Medicine, Hokkaido University, for his kind gift of Vibrio parahaemolyticus Y-4. The authors also thank Dr. Katsutosi Nitta, Department of Polymer, Faculty of Science, Hokkaido University for his advice on circular dichroism measurement.
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