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 ppt

Characterization of isocitrate dehydrogenase from the green sulfur bacterium Chlorobium limicola 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 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 bee n well studied as a catabolic enzyme in the tricarboxylic acid (TCA) cycle, here w e have characterized NADP-dependent IDH from Chlorobium limicola, a green sulfur bacterium that fixes CO 2 through the reductive tricarboxylic acid (RTCA) cycle, focusing on the CO 2 -fixation ability of the enz yme. 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 8 0 kDa distinct from the dimeric NADP-dependent IDHs predominantly found in bacteria or eukaryotic mitochondria. Apparent Michaelis constants for isocitrate (45 ± 13 l M ) and NADP + (27 ± 10 l M ) w ere much smaller than those for 2-oxoglut- arate (1.1 ± 0.5 m M )andCO 2 (1.3 ± 0.3 m M ). No signif- icant diff erences in kinetic properties were observed between Cl-IDH and the dimeric, NADP-dependent IDH from Saccharomyces cerev isiae (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 CO 2 -fixation enzyme under physiological pH. Furthermore, we found that among various intermediates, oxaloacetate was a compe titive inhibitor (K i ¼ 0.35 ± 0.04 m M )for2- oxoglutarate in the carboxylation reaction by Cl-IDH, a feature not found in Sc-IDH. Keywords: isocitrate dehydrogenase; reductive tricarboxylic acid cycle; CO 2 -fixing enzyme. The r eductive tricarboxylic acid (RTCA) cycle is a carbon dioxide (CO 2 ) 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 C O 2 are fixed t o produce one molecule of oxaloacetate in one cycle. It has bee n suggested that the RTCA cycle functions in anaerobic b acteria 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 CO 2 -fixing enzymes: pyruvate synthase, phosphoenolpyruvate carboxylase, 2-oxoglutarate synthase, and isocitrate dehydrogenase (IDH). As I DH is not specific for the RTCA cycle and is 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 aero bic bacteria [ 5], f acultative anaerobic bacteria [6], archaea [7], yeast [8], plants [9], and mammalian tissues [10] [11]. 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 t he 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 k inds of IDH with different cofactor dependency, NAD- and NADP-dependent IDHs. Eukary- otes possess both IDH isozymes, where NAD-dependent enzymes a re a 4 b 4 heterooctamers localized in mitochond ria to function in the T CA cycle, while NADP-dependent IDH activities have been detected in th e cytosol, peroxisomes, and mitochondria. It has been suggested that the eukaryotic NADP-IDHs provide NADPH and 2-oxoglutarate for biosynthesis of fatty a cids and a mino acids [9]. I n 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: + 8 1 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 C hlorobium 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 1 1 December 200 1, revised 1 8 February 2 002, accepted 20 February 2002) Eur. J. Biochem. 269, 1926–1931 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02849.x bacteria possess only NADP-dependent IDH. These bacte- rial and e ukaryotic 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 m onomeric IDHs with molecular masses of  80 kDa have been identified from Azotobacter vinelandii [16], Vibrio parahaemolyticus [17], Rhodomicrobium vannielii [18], Desulfobacter vibrioformis [19], a nd 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]. Th e genes of monomeric IDHs have b een cloned and sequenced from Vibrio sp. ABE-1 [22] and Cr. glutamicum [23], and putative m onomeric IDH genes have been identified on t he chromosomes of Chlorobium tepidum, Pseudomonas aeru- ginosa, Mycobacterium leprae,andNeisseria meningitidis. Comparison of the primary structures revealed little overall similarity between these two types of NADP- dependent IDHs [20] [23]. We have previously isolated the green sulfur bacterium Chlorobium limicola strain M1, and have characterized one of the key enzymes o f the RTC A cycle, ATP-citrate lyase [24]. The results demonstrated the heteromeric structure o f this enzyme and its role in regulating the d irection and flux of the RTCA cycle. For further understanding of the R TCA 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. Furthermor e, the catalytic properties of IDH for the redu ctive carboxylation are much less studied in comparison with those f or the o xidative reaction. In this report, we isolated the gene encoding IDH from C. limicola (Cl-IDH) and characterized the r ecombinant Cl-IDH as a CO 2 -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. MATERIALS AND METHODS Bacteria, plasmids, and media The green sulfur bacterium C. limicola strain M1 was grown phototrophically at 30 °C as described previously [24]. E. coli DH5a and pUC118 were used for DNA manipu- lation and sequ encing. E. coli BL21(DE3) (Stratagene, La Jolla, CA, USA) was used as a host f or an expression plasmid derived from pET21a(+) (Novagen, Madison, WI, USA). T hese strains were cultivated in Luria–Bertani medium at 37 °C. When neces sary, 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, S:G/C, R:A/G) was designed from HLKATMM from position 251–257, and the other primer (5¢-AAYTGYTG NACRTGYTTNGGNGC-3¢) w as a complementary sequence of M AQKAEE from position 409–415 in mono- meric IDH from Cr. glutamicum, respective ly. 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 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 a nd a Model 310 capillary DNA sequencer (Applied Biosystems, Foster City, CA, USA). The multiple alignment o f 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. limicola idh gene and purification of the recombinant enzyme In order to c onstruct an expression vector for Cl -idh,two oligonucleotides (sense, 5¢-A AAAA CATATGGCAAGCA AATCGACCATCATCTACAC-3¢, and antisense, 5¢-AAA AA GGATCCCGGCTGAAAACCGGGCTGCATTA-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 °C. Expression of idh in the recombinant cells under the control o f T7 promoter was induced for 3 h a t 3 7 °C a fter the addition o f 0 .1 m M isopropyl thio-b- D -galactoside (IPTG) when D 660 ¼ 0.4. The cells harvested from a 3-L culture were washed twice with 0.1 M potassium phosphate buffer ( pH 7.2), a nd resuspended in t he 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) b y using an A ¨ KTA explorer 10S apparatus (Amersham Pharmacia Biotech). After equilibrating and washing with 20 m M 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 mLÆmi n )1 . The active fraction was concentrated and further applied onto a Superdex200 HR10/30 g el-filtration column (Amersham Pharmacia Ó FEBS 2002 Isocitrate dehydrogenase from Chlorobium limicola (Eur. J. Biochem. 269) 1927 Biotech) at a flow rate of 0.35 mL Æmin )1 . All purification steps were carried out at 4 °C . The active fractions were examined for ap parent 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 Activities of Cl-IDH and NADP-dependent dimeric IDH from S. cerevisiae (Oriental Yeast, Osaka, Japan) were determined s pectrophotometrically at 25 °C . In the d ecarb - oxylic reaction, the assay mixture contained 0 .4 m M triso- dium DL -isocitrate, 0.2 m M NADP + ,40m M MgCl 2 ,and enzyme solution in 1 m L of 100 m M 2-(cyclohexylamino)- ethanesulfonic acid (Ches) buffer (pH 9.0). T he 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 m M sodium 2-oxoglutarate, 0.16 m M NADPH, 40 m M MgCl 2 ,35m M NaHCO 3 , and enzyme solution in 1 mL of 100 m M N-2-hydroxyethylpiperazine- N¢-2-ethanesulfonic a cid ( Hepes) buffer (pH 7.0). I n order to accurately quantify NaHCO 3 ,0.5 M stock solution of NaHCO 3 in the buffer was preincubated for 1 h before use in an adequately sealed bottle to avoid equilibration with atmospheric CO 2 . After addition of the NaHCO 3 stock solution to the reaction mixture in a s ealed cuvette, further incubation at 25 °C for 5 min was carried out for equili- bration before a ddition o f t he enzyme solution. The 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 f rom 8 .7 to 10.0 were used for the assay. RESULTS Isolation of the idh gene from C. limicola In the cell-free extract of C. limic ola strain M1, we could detect NADP-dependent IDH a ctivity toward isocitrate with a specific activity of 0.85 UÆmg )1 , a s previously shown in the c losely re lated g reen 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 t he 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 c omplete idh gene was isolated from C. limicola genomic library by using the amplified fragment as a p robe. DNA s equencing analysis r evealed that t he Cl-idh gene consisted of 2226-bp and encoded a protein w ith a molecular m ass of 80 465 Da. Putative r ho-independent terminator was located 27-bp downstream of the stop codon. However, typical consensus sequences for ribosome binding and f or a p romoter w ere not identified in the 5¢-flanking region of Cl-idh. No open reading frames were found in the immediate vicinity of the gene. 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 f rom Cr. glutamicum had been expected to be a proton donor during the decarboxylation o f 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 Y 345 were interacted with 2¢-phosphate of NADP molecule [23] and the positively charged residues were highly con served in monomeric IDHs and supposed to contribute t o t heir high specificity toward NADPH. K589 and H590 i n Cl-IDH were proposed to be equivalent to t he residues in Ec -IDH. Expression and purification of IDH from recombinant E. coli A high level of NADP-dependent IDH activity c ould be detected in the cell-free extract after ind uction 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 p rotein was a nalyzed 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 k Da by native- PAGE. Th e 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. Kinetic properties, pH profiles of Cl -IDH and comparison with NADP-dependent IDH from S. cerevisiae The catalytic properties of IDH from C. limicola were investigated for both the oxidative decarboxylation and reductive carboxylation r eactions. The activity for oxidative decarboxylation of isocitrate was assayed by standard procedures. The optimum pH was 9.0 (Fig. 2A), and a pparent K m values for isocitrate a nd NADP + at the optimum pH were determined to be 45 ± 13 l M and 27 ± 10 l M , respectively (Table 2). The reductive carboxy- lation activity towards 2-oxoglutarate was determined also by spectrophotometry. As both the monomeric and d imeric IDHs have been reported to accept CO 2 molecule as a substrate [27] [28], the reaction mixture was sufficiently equilibrated after addition of NaHCO 3 solution prior to assay in a capped c uvette. The optimum pH for carboxy- lation was 7 .0 (Fig. 2A), where the C O 2 concentration after the equilibration was 17.9% (6.27 m M ) of initial bicarbonate concentration (35 m M ). Under t his reaction condition, Cl-IDH showed normal M ichaelis–Menten k inetics also 1928 T. Kanao et al. (Eur. J. Biochem. 269) Ó FEBS 2002 for the carboxylation reaction. Apparent K m values for 2-oxoglutarate and CO 2 were 1.1 ± 0.5 m M and 1.3 ± 0.3 m M , respectively, which were much greater than the values f or isocitra te and N ADP + . The kinetic param- eters of monomeric IDHs from C. limicola and A. vinelan- dii, p reviously determined by Wicken et al. [28], are also shown in Table 2. In addition, we further examined the catalytic properties of NADP-dependent IDH f rom 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 K m values for isocitrate, NADP + , 2-oxoglutarate, and CO 2 were 20 ± 5 l M ,33±6l M ,0.85±0.30m M , and 8.2 ± 1.0 m M , respectively (Table 2). The results suggested that differe nces 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 c arboxylic activities of Cl-IDH ( 46.0 and 41.0 U Æmg )1 , r espectively) were almost equivalent under physiological conditions (Fig. 2A), in contrast to the m uch higher activity for d ecarboxylation o f Sc-IDH (41.0 UÆmg )1 ) than t hat f or car boxylation (8.7 UÆmg )1 )at pH 7 .0 (Fig. 2B). Fig. 1. Native-PAGE of recombinant Cl-IDH. The a ctive 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 00 0 Da), a lbum in (66 0 00 Da). Fig. 2. Effect of pH on the de carboxylation (open symbols) and carb- oxylation (closed symbols) activities of Cl-IDH (A) and Sc-IDH (B). Assays were performed i n e ach buffer as follows; Mes (r,e), Hepes (j,h), Bicine ( m,n), CHES (d,s). Table 2. Comparison o f kinetic pro perties of ID Hs. Cl, Chlorobiu m limico la ; Sc, Saccharomyces cerevisiae; Av, Azotobacter vinelandii. Reaction Properties Cl-IDH Sc-IDH Av-IDH (28) Decarboxylation K m (l M ) Isocitrate 45 ± 13 20 ± 5 6.8 NADP 27 ± 10 33 ± 6 8.3 V max (UÆmg )1 ) 150 ± 6 54 ± 5 130 Carboxylation K m (m M ) 2-Oxoglutarate 1.1 ± 0.5 0.85 ± 0.30 0.0139 CO 2 1.3 ± 0.3 8.2 ± 1.0 0.39 V max (UÆmg )1 ) 38±9 16±2 – Table 1. Purification o f Cl-IDH from recombinant E. co li. IDH activity was measured with carboxylation reaction. Step Total protein (mg) Total activity (U) Specific activity (UÆmg )1 ) Yield (%) Purification (fold) Cell-free extract 254 4950 19.5 100 1 ResourceQ 100 2640 26.4 53.3 1.35 Superdex200 58.4 2100 36.0 42.5 1.84 Ó FEBS 2002 Isocitrate dehydrogenase from Chlorobium limicola (Eur. J. Biochem. 269) 1929 Inhibition of carboxylation activity of Cl -IDH by oxaloacetate We further examined the effects of intermediate compounds in the RTCA cycle on Cl -IDH ac tivity. C itrate, p yruvate, succinate, fumarate, m alate, glyoxylate, ATP, and ADP gave no significant effect on the carboxylation reaction (data not shown). H owever, considerable inhibition was observed when oxaloacetate was added into t he mixture. The carboxylation activity decreased to more t han half in the presence of 1 m M oxaloacetate (Fig. 3 A). In contrast, up to 5 m M oxaloacetate had no influence on the carboxylation activity of Sc-IDH. The Dixon plot for o xaloacetate with different concentrations of 2-oxoglutarate displayed typical competitive inhibition, and a K i value of oxaloacetate for Cl-IDH was determined to be 0.35 ± 0.04 m M (Fig. 3 B). Similar to previous reports with IDHs from various sources, a concerted inhibition with oxaloacetate a nd glyoxylate was also observed against Cl-IDH. By a ddition of 0.25 m M glyoxylate t ogether with the same concentration o f oxaloacetate (0.25 m M ), the decarboxylation activity was decreased to 44%, w hile relative activity was 77% wit hout glyoxylate (data not s hown). DISCUSSION In this paper, we investigated IDH from the green sulfur bacterium, C. limicola,asaCO 2 -fixing enzyme in RTCA cycle. The enzyme IDH from C. limicola was revealed t o 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. g lutamicum. However, no signi ficant similarity was observed between monomeric and dimeric IDHs in their primary structures, suggesting that t hese two distinct IDHs evolved independently from different ances- tors. We compared the catalytic properties of Cl -IDH with those o f Sc-IDH. Both IDHs exhibited higher affinities to substrates for decarboxylation (NADP + and isocitrate) than those for carboxylation (2-oxoglutarate and CO 2 ), and the specific activities toward decarboxylation were higher than those toward the reverse direction at the respective optimum pH. The kinetic parameters of monomeric I DH 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 oxidat ive decarboxylation m ore 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 f or reduction of unsaturated f atty acid in S. cerevisiae [29]. In contrast, the carboxylation activity o f Cl-IDH at pH 7.0 was as high as the decarboxylation activity (Fig. 2 A). Cl-IDH possessed a more favorable property to fix CO 2 than Sc-IDH under physiological conditions. Among the intermediates of RTCA cycle, oxaloacetate affected activities of Cl-IDH. More than half of the activity was i nhibited by 1 m M oxaloacetate in both d irections, and the inhibition for carboxylation was shown to be compet- itive. Inhibition by oxaloacetate has been examined for dimeric IDHs and a few monomeric IDHs fr om Cr. g lutamicum and A. vinelandii. The enzymes displayed low (5–27%), or only trivial (0–5%) levels of inhibition by 1m M oxaloacetate. Although these results were obtained against d ecarboxylic activi ty, w e confirmed that even 5 m M oxaloacetate gave no inhibition to the carboxylation activity of Sc-IDH (Fig. 3A). IDHs seemed to be gene rally inse n- sitive against oxaloacetate. One exception is the I DH from purple nonsulfur bacterium R. vannielii, which showed 44% inhibition with 0.2 m M 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. 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 p ossibilities of oxaloacetate accumulation. Indeed, when we have analyzed the malate dehydrogenase a ctivity in the cell-free extracts of C. limico- la,0.95UÆmg )1 activity in the direction o f m alate synthesis could be detected (data not shown). However, a lthough a closely related strain Chlorobium thiosulfatophilum also harbors the same levels of malate dehydrogenase (0.62 UÆmg )1 [1]), previous radiolabeling experiments dem- onstrated a large accumulation of oxaloacetate in the cells, relative to other intermediates [30]. In the cells of C. thiosulfatophilum grown i n a medium containing 3 H 2 O, the radioactivity of oxaloacetate was 3.6-fold greater t han that of malate and 21-fold greater t han that of the sum of citrate and isocitrate. I n addition, 14 CO 2 -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 t he concen tration w ould s ensitively refle ct the level of carbon assimilation by the cycle. As we previously repo rted, ATP-citrate lyase from C. limic ola catalyzes only t he Fig. 3. Inhibition of Cl -IDH with oxaloacetate. (A) E ffect of oxaloac- etate concentration on the carboxylation activities of Cl-IDH (j)and Sc-IDH ( h). (B ) Dixon-plots for oxaloacetate w ith 3 m M (d), 5 m M (m), and 1 0 m M (s) 2-oxoglutarate. 1930 T. Kanao et al. (Eur. J. Biochem. 269) Ó FEBS 2002 ATP-dependent cleavage of citrate and the activity w as 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 t o overaccumulation of oxaloacetate within the cells. 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