Báo cáo khoa học: Characterization of isocitrate dehydrogenase from the green sulfur bacterium Chlorobium limicola A carbon dioxide-fixing enzyme in the reductive tricarboxylic acid cycle

doi:10.1046/j.1432-1033.2002.02849.x

Eur. J. Biochem. 269, 1926–1931 (2002) (cid:211) FEBS 2002

Characterization of isocitrate dehydrogenase from the green sulfur bacterium Chlorobiumlimicola A carbon dioxide-fixing enzyme in the reductive tricarboxylic acid cycle

Tadayoshi Kanao, Mineko Kawamura, Toshiaki Fukui, Haruyuki Atomi and Tadayuki Imanaka

Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto, Japan

arate (1.1 (cid:139) 0.5 mM) and CO2 (1.3 (cid:139) 0.3 mM). No signif- icant differences in kinetic properties were observed between Cl-IDH and the dimeric, NADP-dependent IDH from Saccharomyces cerevisiae (Sc-IDH) at the optimum pH of each enzyme. However, in contrast to the 20% activity of Sc-IDH toward carboxylation as compared with that to- ward decarboxylation at pH 7.0, the activities of Cl-IDH for both directions were almost equivalent at this pH, suggesting a more favorable property of Cl-IDH than Sc-IDH as a CO2-fixation enzyme under physiological pH. Furthermore, we found that among various intermediates, oxaloacetate was a competitive inhibitor (Ki (cid:136) 0.35 (cid:139) 0.04 mM) for 2- oxoglutarate in the carboxylation reaction by Cl-IDH, a feature not found in Sc-IDH.

for

Keywords: isocitrate dehydrogenase; reductive tricarboxylic acid cycle; CO2-fixing enzyme.

Isocitrate dehydrogenase (IDH) catalyzes the reversible conversion between isocitrate and 2-oxoglutarate accom- panied by decarboxylation/carboxylation and oxidoreduc- tion of NAD(P)+ cofactor. While this enzyme has been well studied as a catabolic enzyme in the tricarboxylic acid (TCA) cycle, here we have characterized NADP-dependent IDH from Chlorobium limicola, a green sulfur bacterium that fixes CO2 through the reductive tricarboxylic acid (RTCA) cycle, focusing on the CO2-fixation ability of the enzyme. The gene encoding Cl-IDH consisted of 2226 bp, corresponding to a polypeptide of 742 amino acid residues. The primary struc- ture and the size of the recombinant protein indicated that Cl-IDH was a monomeric enzyme of 80 kDa distinct from the dimeric NADP-dependent IDHs predominantly found in bacteria or eukaryotic mitochondria. Apparent Michaelis isocitrate (45 (cid:139) 13 lM) and NADP+ constants (27 (cid:139) 10 lM) were much smaller than those for 2-oxoglut-

widely distributed as a member of the tricarboxylic acid (TCA) cycle, this enzyme has been extensively characterized in terms of its contribution to the TCA cycle in various species, including aerobic bacteria [5], facultative anaerobic bacteria [6], archaea [7], yeast [8], plants [9], and mammalian tissues [10] [11].

synthase, phosphoenolpyruvate

The reductive tricarboxylic acid (RTCA) cycle is a carbon dioxide (CO2) fixation pathway distinct from the well- known reductive pentose phosphate cycle (Calvin–Benson cycle) in plants, algae, and various bacteria. In this pathway, four molecules of CO2 are fixed to produce one molecule of oxaloacetate in one cycle. It has been suggested that the RTCA cycle functions in anaerobic bacteria Chlorobium [1] and Desulfobacter [2], thermophilic bacteria Hydrogeno- bacter [3] and Aquifex [4], and also in the thermophilic archaeon Thermoproteus [4]. The key enzymes of the RTCA cycle are ATP-citrate lyase, and four CO2-fixing enzymes: pyruvate carboxylase, 2-oxoglutarate synthase, and isocitrate dehydrogenase (IDH). As IDH is not specific for the RTCA cycle and is

IDH in the TCA cycle catalyzes the oxidative decarb- oxylation of isocitrate to 2-oxoglutarate coupled with the reduction of NAD(P)+. The IDH reaction is not only an oxidation step in the cycle for generation of reducing power but also provides 2-oxoglutarate as an important inter- mediate for glutamate biosynthesis. Indeed, deficiency of this enzyme in Escherichia coli resulted in the auxotrophy for glutamate [12]. IDH also comprises the branching point between TCA cycle and glyoxylate cycle along with isocitrate lyase. In E. coli and related bacteria grown on C2 carbon sources, IDH is phosphorylated by the function of IDH kinase/phosphatase, that leads to inactivation of the enzyme and consequent switch of the carbon flux from TCA cycle to glyoxylate cycle [13] [14].

There are two kinds of IDH with different cofactor dependency, NAD- and NADP-dependent IDHs. Eukary- otes possess both IDH isozymes, where NAD-dependent enzymes are a4b4 heterooctamers localized in mitochondria to function in the TCA cycle, while NADP-dependent IDH activities have been detected in the cytosol, peroxisomes, and mitochondria. It has been suggested that the eukaryotic NADP-IDHs provide NADPH and 2-oxoglutarate for biosynthesis of fatty acids and amino acids [9]. In contrast,

Correspondence to T. Imanaka, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501, Japan. Fax: + 81 75 7534703, Tel.: + 81 75 7535568, E-mail: imanaka@sbchem.kyoto-u.ac.jp Abbreviations: RTCA cycle, reductive tricarboxylic acid cycle; IDH, isocitrate dehydrogenase; idh, the gene encoding isocitrate dehydro- genase; Cl-IDH, isocitrate dehydrogenase from Chlorobium limicola; Sc-IDH, NADP-dependent isocitrate dehydrogenase from Sacchar- omyces cerevisiae; IPTG, isopropyl thio-b-D-galactoside. Enzyme: isocitrate dehydrogenase (EC 1.1.1.42). (Received 11 December 2001, revised 18 February 2002, accepted 20 February 2002)

Isocitrate dehydrogenase from Chlorobium limicola (Eur. J. Biochem. 269) 1927

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a

vinelandii

[17], Rhodomicrobium vannielii

vibrioformis

S:G/C, R:A/G) was designed from HLKATMM from position 251–257, and the other primer (5¢-AAYTGYTG NACRTGYTTNGGNGC-3¢) was complementary sequence of MAQKAEE from position 409–415 in mono- meric IDH from Cr. glutamicum, respectively. A phage clone carrying the complete Cl-idh gene was screened from the genomic library by plaque hybridization using the amplified DNA fragment as a probe. A BamHI and SalI restriction fragment containing the idh gene and its flanking regions (6.0 kbp) was subcloned into pUC118.

DNA manipulation and sequencing

bacteria possess only NADP-dependent IDH. These bacte- rial and eukaryotic NADP-dependent IDHs are usually dimeric in structure, consisting of identical subunits with molecular masses ranging from 40 to 57 kDa [9] [15]. In addition to these enzymes, a limited number of monomeric IDHs with molecular masses of (cid:25) 80 kDa have been [16], Vibrio identified from Azotobacter parahaemolyticus [18], Desulfobacter [19], and Corynebacterium glutamicum [20]. Psychrophilic Vibrio sp. strain ABE-1 possesses structurally distinct IDH isozymes of homodi- meric (IDH-I) and monomeric (IDH-II) structures [21]. The genes of monomeric IDHs have been cloned and sequenced from Vibrio sp. ABE-1 [22] and Cr. glutamicum [23], and putative monomeric IDH genes have been identified on the chromosomes of Chlorobium tepidum, Pseudomonas aeru- ginosa, Mycobacterium leprae, and Neisseria meningitidis. Comparison of the primary structures revealed little overall similarity between these two types of NADP- dependent IDHs [20] [23].

DNA manipulation was carried out according to the methods described by Sambrook & Russell [25]. Prepara- tion of plasmid DNA was performed with Plasmid Mini- and Midi-Kits (Qiagen, Hilden, Germany) along with the alkaline extraction method [25]. Nucleotide sequences of both DNA strands were determined using a BigDye Terminator Cycle Sequencing FS Ready Reaction Kit and a Model 310 capillary DNA sequencer (Applied Biosystems, Foster City, CA, USA). The multiple alignment of protein sequences and the identity and similarity between sequences were obtained with the program ALIGN contained within the CLUSTALW program provided by DNA Data Bank of Japan (DDBJ). The sequence data was analyzed using GENETYX software package (Software Development, Tokyo, Japan). The nucleotide sequence data of Cl-idh will appear in the EMBL, GenBank, and DDBJ nucleotide sequence data- bases under accession no. AB076021.

Expression of C.limicolaidh gene and purification of the recombinant enzyme

We have previously isolated the green sulfur bacterium Chlorobium limicola strain M1, and have characterized one of the key enzymes of the RTCA cycle, ATP-citrate lyase [24]. The results demonstrated the heteromeric structure of this enzyme and its role in regulating the direction and flux of the RTCA cycle. For further understanding of the RTCA cycle, we are carrying out detailed investigations of each member of the cycle. With respect to IDH, although the activities have been detected in some autotrophic organisms utilizing the RTCA cycle, no biochemical analysis of the enzyme has been reported. Furthermore, the catalytic properties of IDH for the reductive carboxylation are much less studied in comparison with those for the oxidative reaction.

In this report, we isolated the gene encoding IDH from C. limicola (Cl-IDH) and characterized the recombinant Cl-IDH as a CO2-fixing enzyme, and compared the catalytic properties of Cl-IDH with those of dimeric NADP-depen- dent IDH from Saccharomyces cerevisiae having different physiological functions.

M A T E R I A L S A N D M E T H O D S

Bacteria, plasmids, and media

The green sulfur bacterium C. limicola strain M1 was grown phototrophically at 30 (cid:176)C as described previously [24]. E. coli DH5a and pUC118 were used for DNA manipu- lation and sequencing. E. coli BL21(DE3) (Stratagene, La Jolla, CA, USA) was used as a host for an expression plasmid derived from pET21a(+) (Novagen, Madison, WI, USA). These strains were cultivated in Luria–Bertani medium at 37 (cid:176)C. When necessary, 50 lgÆmL)1 ampicillin was supplied into the medium to maintain plasmids.

Isolation of the IDH gene (idh) from C.limicola

Construction of a genomic DNA library of C. limicola M1 has been described previously [24]. A partial DNA fragment of idh was amplified from C. limicola genomic DNA by PCR using two primers corresponding to highly conserved regions among monomeric IDHs. One primer (5¢-CAYC TSAARGCNACSATGATG-3¢, N:A/T/G/C, Y:C/T,

In order to construct an expression vector for Cl-idh, two oligonucleotides (sense, 5¢-AAAAACATATGGCAAGCA AATCGACCATCATCTACAC-3¢, and antisense, 5¢-AAA AAGGATCCCGGCTGAAAACCGGGCTGCATTA-3¢) were designed for amplification of idh flanked with NdeI and BamHI sites (underlined). After confirming the nucle- otide sequence, an NdeI–BamHI fragment of the amplified idh gene was ligated with pET21a(+) at the corresponding sites. The expression vector, named pET-IDH, was intro- duced into E. coli BL21(DE3), and the recombinant cells were cultured in Luria–Bertani medium containing 50 lgÆmL)1 ampicillin at 37 (cid:176)C. Expression of idh in the recombinant cells under the control of T7 promoter was induced for 3 h at 37 (cid:176)C after the addition of 0.1 mM isopropyl thio-b-D-galactoside (IPTG) when D660 (cid:136) 0.4. The cells harvested from a 3-L culture were washed twice with 0.1 M potassium phosphate buffer (pH 7.2), and resuspended in the same buffer. The cells were disrupted by sonication on ice, and then centrifuged for 15 min at 15 000 g to remove cell debris. The soluble fraction was applied onto a Resource Q anion exchange column (Amer- sham Pharmacia Biotech, Uppsala, Sweden) by using an A¨ KTA explorer 10S apparatus (Amersham Pharmacia Biotech). After equilibrating and washing with 20 mM potassium phosphate buffer (pH 7.2), Cl-IDH was eluted by a linear gradient of KCl (0–0.5 M) in the same buffer with a flow rate of 2 mLÆmin)1. The active fraction was concentrated and further applied onto a Superdex200 HR10/30 gel-filtration column (Amersham Pharmacia

1928 T. Kanao et al. (Eur. J. Biochem. 269)

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terminator was located 27-bp downstream of the stop codon. However, typical consensus sequences for ribosome binding and for a promoter were not identified in the 5¢-flanking region of Cl-idh. No open reading frames were found in the immediate vicinity of the gene.

Biotech) at a flow rate of 0.35 mLÆmin)1. All purification steps were carried out at 4 (cid:176)C. The active fractions were examined for apparent homogeneity by SDS/PAGE. Pro- tein concentration was determined by a Bio-Rad Protein Assay system (Bio-Rad, Hercules, CA, USA) with bovine serum albumin as a standard.

Enzyme assays

The deduced amino-acid sequence of Cl-IDH was 66.0% and 57.4% identical to monomeric IDHs from Vibrio sp. strain ABE-1 (IDH-II) and from Cr. glutamicum, respect- ively. K253 in IDH from Cr. glutamicum had been expected to be a proton donor during the decarboxylation of isocitrate, and indeed, the site-specific mutagenesis of K253 to Met led to an inactive protein [23]. In addition, the alkylation of the adjacent M258 inactivated the IDH from A. vinelandii [26]. These Lys and Met residues were conserved in Cl-IDH at the position of 256 and 259, respectively. In dimeric IDH from E. coli (Ec-IDH), K344 and Y345 were interacted with 2¢-phosphate of NADP molecule [23] and the positively charged residues were highly conserved in monomeric IDHs and supposed to contribute to their high specificity toward NADPH. K589 and H590 in Cl-IDH were proposed to be equivalent to the residues in Ec-IDH.

Expression and purification of IDH from recombinant E.coli

Activities of Cl-IDH and NADP-dependent dimeric IDH from S. cerevisiae (Oriental Yeast, Osaka, Japan) were determined spectrophotometrically at 25 (cid:176)C. In the decarb- oxylic reaction, the assay mixture contained 0.4 mM triso- dium DL-isocitrate, 0.2 mM NADP+, 40 mM MgCl2, and enzyme solution in 1 mL of 100 mM 2-(cyclohexylamino)- ethanesulfonic acid (Ches) buffer (pH 9.0). The increase of NADPH was detected by absorbance at 340 nm, and one unit of activity was defined as 1 lmol of NADPH formed per min. In the carboxylation reaction, the mixture was composed of 8 mM sodium 2-oxoglutarate, 0.16 mM NADPH, 40 mM MgCl2, 35 mM NaHCO3, and enzyme solution in 1 mL of 100 mM N-2-hydroxyethylpiperazine- N¢-2-ethanesulfonic acid (Hepes) buffer (pH 7.0). In order to accurately quantify NaHCO3, 0.5 M stock solution of NaHCO3 in the buffer was preincubated for 1 h before use in an adequately sealed bottle to avoid equilibration with atmospheric CO2. After addition of the NaHCO3 stock solution to the reaction mixture in a sealed cuvette, further incubation at 25 (cid:176)C for 5 min was carried out for equili- the enzyme solution. The bration before addition of consumption of NADPH was monitored at 340 nm, and one unit of activity was defined as 1 lmol of NADPH oxidized per min. For determination of optimum pH in each reaction, 2-(N-morpholino)ethanesulfonic acid (Mes) buf- fers with pH values from 5.0 to 7.0, Hepes buffers with pH values from 7.0 to 8.5, N,N-bis(2-hydroxyethyl)glycine (Bicine) buffers with pH values from 8.0 to 9.0, and Ches buffers with pH values from 8.7 to 10.0 were used for the assay.

A high level of NADP-dependent IDH activity could be detected in the cell-free extract after induction with IPTG. The activity of the recombinant cell-extract (19.5 UÆmg)1) was 100-fold higher than that in the host cells (0.20 UÆmg)1). The homogeneity of the recombinant protein was analyzed with SDS/PAGE (data not shown) and native-PAGE (Fig. 1) analyses, and the specific activity of the purified IDH reached 36.0 UÆmg)1 (Table 1). The molecular mass of the native enzyme was determined to be 81 kDa by gel- filtration column chromatography and 80 kDa by native- PAGE. The results indicated that the recombinant IDH was a monomeric enzyme with a molecular mass of 80 kDa. No IDH activity was detected when NADH was used as a cofactor.

R E S U L T S

Isolation of the idhgene from C.limicola

Kinetic properties, pH profiles of Cl-IDH and comparison with NADP-dependent IDH from S.cerevisiae

In the cell-free extract of C. limicola strain M1, we could detect NADP-dependent IDH activity toward isocitrate with a specific activity of 0.85 UÆmg)1, as previously shown in the closely related green sulfur bacterium, C. thiosulfato- philum [1]. Steen et al. have recently reported the presence of IDH in a related thermophile C. tepidum by activity staining after SDS/PAGE, in which the active band corresponded in size (80 kDa) to monomeric IDH from Desulfobacter vibrioformis [19]. We therefore supposed that IDH from C. limicola is likely to be a monomeric enzyme. Two primers were designed from conserved regions among known monomeric IDHs (See Materials and methods), and PCR with the primers and genomic DNA from strain M1 gave successful amplification of a 1-kbp DNA fragment. The complete idh gene was isolated from C. limicola genomic library by using the amplified fragment as a probe. DNA sequencing analysis revealed that the Cl-idh gene consisted of 2226-bp and encoded a protein with a molecular mass of 80 465 Da. Putative rho-independent

The catalytic properties of IDH from C. limicola were investigated for both the oxidative decarboxylation and reductive carboxylation reactions. The activity for oxidative decarboxylation of isocitrate was assayed by standard procedures. The optimum pH was 9.0 (Fig. 2A), and apparent Km values for isocitrate and NADP+ at the optimum pH were determined to be 45 (cid:139) 13 lM and 27 (cid:139) 10 lM, respectively (Table 2). The reductive carboxy- lation activity towards 2-oxoglutarate was determined also by spectrophotometry. As both the monomeric and dimeric IDHs have been reported to accept CO2 molecule as a substrate [27] [28], the reaction mixture was sufficiently equilibrated after addition of NaHCO3 solution prior to assay in a capped cuvette. The optimum pH for carboxy- lation was 7.0 (Fig. 2A), where the CO2 concentration after the equilibration was 17.9% (6.27 mM) of initial bicarbonate concentration (35 mM). Under this reaction condition, Cl-IDH showed normal Michaelis–Menten kinetics also

Isocitrate dehydrogenase from Chlorobium limicola (Eur. J. Biochem. 269) 1929

(cid:211) FEBS 2002

Fig. 2. Effect of pH on the decarboxylation (open symbols) and carb- oxylation (closed symbols) activities of Cl-IDH (A) and Sc-IDH (B). Assays were performed in each buffer as follows; Mes (r,e), Hepes (j,h), Bicine (m,n), CHES (d,s).

decarboxylation

activity

for

of

and CO2 were

the values for isocitrate and NADP+. The kinetic param- eters of monomeric IDHs from C. limicola and A. vinelan- dii, previously determined by Wicken et al. [28], are also shown in Table 2. In addition, we further examined the catalytic properties of NADP-dependent IDH from S. cerevisiae (Sc-IDH) in order to compare the properties of monomeric IDHs with those of a dimeric enzyme. The optimum pH for the decarboxylation and carboxylation activities of Sc-IDH were 8.5 and 6.0 (Fig. 2B), and apparent Km values for isocitrate, NADP+, 2-oxoglutarate, and CO2 were 20 (cid:139) 5 lM, 33 (cid:139) 6 lM, 0.85 (cid:139) 0.30 mM, and 8.2 (cid:139) 1.0 mM, respectively (Table 2). The results suggested that differences in the properties were not so significant among the three enzymes for both the directions. However, an interesting difference between Cl-IDH and Sc-IDH was observed in the activities at pH 7.0. The decarboxylic and carboxylic activities of Cl-IDH (46.0 and 41.0 UÆmg)1, respectively) were almost equivalent under physiological conditions (Fig. 2A), in contrast to the much Sc-IDH higher (41.0 UÆmg)1) than that for carboxylation (8.7 UÆmg)1) at pH 7.0 (Fig. 2B).

for the carboxylation reaction. Apparent Km values for 1.1 (cid:139) 0.5 mM and 2-oxoglutarate 1.3 (cid:139) 0.3 mM, respectively, which were much greater than

Fig. 1. Native-PAGE of recombinant Cl-IDH. The active fraction after Superdex200 gel-filtration column chromatography was applied to lane 1. Lane M, molecular markers, thyroglobulin (669 000 Da), ferritin (440 000 Da), catalase (232 000 Da), lactate dehydrogenase (140 000 Da), albumin (66 000 Da).

Table 1. Purification of Cl-IDH from recombinant E. coli. IDH activity was measured with carboxylation reaction.

Total protein (mg) Total activity (U) Specific activity (UÆmg)1) Yield (%) Purification (fold) Step

Cell-free extract ResourceQ Superdex200 254 100 58.4 4950 2640 2100 19.5 26.4 36.0 100 53.3 42.5 1 1.35 1.84

Table 2. Comparison of kinetic properties of IDHs. Cl, Chlorobium limicola; Sc, Saccharomyces cerevisiae; Av, Azotobacter vinelandii.

Reaction Properties Cl-IDH Sc-IDH Av-IDH(28)

Decarboxylation Km (lM)

45 (cid:139) 13 27 (cid:139) 10 150 (cid:139) 6 20 (cid:139) 5 33 (cid:139) 6 54 (cid:139) 5 6.8 8.3 130 Carboxylation Isocitrate NADP Vmax (UÆmg)1) Km (mM)

2-Oxoglutarate CO2 1.1 (cid:139) 0.5 1.3 (cid:139) 0.3 38 (cid:139) 9 0.85 (cid:139) 0.30 8.2 (cid:139) 1.0 16 (cid:139) 2 0.0139 0.39 – Vmax (UÆmg)1)

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from A. Vinelandii also showed the same tendency (Table 2). These results indicated that there was not such a significant difference between Cl-IDH and the counter- parts from aerobic microorganisms. These IDHs could catalyze oxidative decarboxylation more efficiently com- pared to reductive carboxylation.

However,

it is interesting to note that the ratios of carboxylation and decarboxylation activities at pH 7.0, presumably close to the physiological pH, showed a clear difference between Cl-IDH and Sc-IDH. The activity of Sc-IDH toward decarboxylation was fivefold higher than that toward carboxylation at pH 7.0 (Fig. 2B), suggesting that the decarboxylic reaction was predominant over the carboxylic reaction in vivo. This result is consistent with the fact that the NADP-dependent IDH contributes to provide NADPH for reduction of unsaturated fatty acid in S. cerevisiae [29]. In contrast, the carboxylation activity of Cl-IDH at pH 7.0 was as high as the decarboxylation activity (Fig. 2A). Cl-IDH possessed a more favorable property to fix CO2 than Sc-IDH under physiological conditions.

Inhibition of carboxylation activity of Cl-IDH by oxaloacetate

IDHs

IDHs and a few monomeric

Among the intermediates of RTCA cycle, oxaloacetate affected activities of Cl-IDH. More than half of the activity was inhibited by 1 mM oxaloacetate in both directions, and the inhibition for carboxylation was shown to be compet- itive. Inhibition by oxaloacetate has been examined for dimeric from Cr. glutamicum and A. vinelandii. The enzymes displayed low (5–27%), or only trivial (0–5%) levels of inhibition by 1 mM oxaloacetate. Although these results were obtained against decarboxylic activity, we confirmed that even 5 mM oxaloacetate gave no inhibition to the carboxylation activity of Sc-IDH (Fig. 3A). IDHs seemed to be generally insen- sitive against oxaloacetate. One exception is the IDH from purple nonsulfur bacterium R. vannielii, which showed 44% inhibition with 0.2 mM oxaloacetate [18]. This indi- cates that the strong inhibition by oxaloacetate was not a specific property for an IDH which functions in the RTCA cycle.

We further examined the effects of intermediate compounds in the RTCA cycle on Cl-IDH activity. Citrate, pyruvate, succinate, fumarate, malate, glyoxylate, ATP, and ADP gave no significant effect on the carboxylation reaction (data not shown). However, considerable inhibition was observed when oxaloacetate was added into the mixture. The carboxylation activity decreased to more than half in the presence of 1 mM oxaloacetate (Fig. 3A). In contrast, up to 5 mM oxaloacetate had no influence on the carboxylation activity of Sc-IDH. The Dixon plot for oxaloacetate with different concentrations of 2-oxoglutarate displayed typical competitive inhibition, and a Ki value of oxaloacetate for Cl-IDH was determined to be 0.35 (cid:139) 0.04 mM (Fig. 3B). Similar to previous reports with IDHs from various sources, a concerted inhibition with oxaloacetate and glyoxylate was also observed against Cl-IDH. By addition of 0.25 mM glyoxylate together with the same concentration of oxaloacetate (0.25 mM), the decarboxylation activity was decreased to 44%, while relative activity was 77% without glyoxylate (data not shown).

Fig. 3. Inhibition of Cl-IDH with oxaloacetate. (A) Effect of oxaloac- etate concentration on the carboxylation activities of Cl-IDH (j) and Sc-IDH (h). (B) Dixon-plots for oxaloacetate with 3 mM (d), 5 mM (m), and 10 mM (s) 2-oxoglutarate.

D I S C U S S I O N

the

same

[30].

In this paper, we investigated IDH from the green sulfur bacterium, C. limicola, as a CO2-fixing enzyme in RTCA cycle. The enzyme IDH from C. limicola was revealed to be a monomeric enzyme with a molecular mass of 80.5 kDa. The deduced amino-acid sequence of Cl-idh gene showed high similarities to other monomeric IDHs from Vibrio sp. strain ABE-1 and Cr. glutamicum. However, no significant similarity was observed between monomeric and dimeric IDHs in their primary structures, suggesting that these two distinct IDHs evolved independently from different ances- tors.

We compared the catalytic properties of Cl-IDH with those of Sc-IDH. Both IDHs exhibited higher affinities to substrates for decarboxylation (NADP+ and isocitrate) than those for carboxylation (2-oxoglutarate and CO2), and the specific activities toward decarboxylation were higher than those toward the reverse direction at the respective optimum pH. The kinetic parameters of monomeric IDH

The question remains whether Cl-IDH is actually inhib- ited by oxaloacetate in vivo. Malate dehydrogenase is known to predominantly catalyze the reduction of oxaloacetate to malate, and thereby lowering the possibilities of oxaloacetate accumulation. Indeed, when we have analyzed the malate dehydrogenase activity in the cell-free extracts of C. limico- la, 0.95 UÆmg)1 activity in the direction of malate synthesis could be detected (data not shown). However, although a closely related strain Chlorobium thiosulfatophilum also levels of malate dehydrogenase harbors (0.62 UÆmg)1 [1]), previous radiolabeling experiments dem- onstrated a large accumulation of oxaloacetate in the cells, relative to other intermediates In the cells of C. thiosulfatophilum grown in a medium containing 3H2O, the radioactivity of oxaloacetate was 3.6-fold greater than that of malate and 21-fold greater than that of the sum of citrate and isocitrate. In addition, 14CO2-labeling indicated that oxaloacetate was one of the first stable products of photosynthesis by C. thiosulfatophilum [30]. These results suggested that oxaloacetate was pooled in Chlorobium cells despite the presence of high malate dehydrogenase activity, and the concentration would sensitively reflect the level of carbon assimilation by the cycle. As we previously reported, ATP-citrate lyase from C. limicola catalyzes only the

Isocitrate dehydrogenase from Chlorobium limicola (Eur. J. Biochem. 269) 1931

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15. Muro-Pastor, M.I. & Florencio, F.J. (1994) NADP+-isocitrate dehydrogenase from the cyanobacterium Anabaena sp. strain PCC 7120: purification and characterization of the enzyme and cloning, sequencing, and disruption of the icd gene. J. Bacteriol. 176, 2718–2726.

ATP-dependent cleavage of citrate and the activity was inhibited at higher ADP/ATP ratios [24]. RTCA cycle is considered to be driven excessively by ATP-citrate lyase under sufficient energy conditions that might lead to overaccumulation of oxaloacetate within the cells. The inhibition of Cl-IDH carboxylic reaction by oxaloacetate or by that concerted with glyoxylate could suppress the cycle in order to change the carbon flux to other pathways, such as glutamate biosynthesis, under the condition of excess turnover of the RTCA cycle. Further studies of the enzyme will clarify the structure, functions, and regulation in the RTCA cycle.

16. Chung, A.E. & Franzen, J.S. (1969) Oxidized triphosphopyridine nucleotide specific isocitrate dehydrogenase from Azotobacter vinelandii. Isolation and characterization. Biochemistry 8, 3175–3184.

17. Fukunaga, N., Imagawa, S., Sahara, T., Ishii, A. & Suzuki, M. (1992) Purification and characterization of monomeric isocitrate dehydrogenase with NADP+-specificity from Vibrio parahaemo- lyticus Y-4. J. Biochem. 112, 849–855.

R E F E R E N C E S

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