Characterization of isocitrate dehydrogenase from the green sulfurA carbon dioxide-fixing enzyme in the reductive tricarboxylic acid cycle Tadayoshi Kanao, Mineko Kawamura, Toshiaki Fuku
Trang 1Characterization of isocitrate dehydrogenase from the green sulfur
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 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
CO2through 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
constants for isocitrate (45 ± 13 lM) and NADP+
(27 ± 10 lM) were much smaller than those for
2-oxoglut-arate (1.1 ± 0.5 mM) and CO2(1.3 ± 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 ¼ 0.35 ± 0.04 mM) for 2-oxoglutarate in the carboxylation reaction by Cl-IDH, a feature not found in Sc-IDH
Keywords: isocitrate dehydrogenase; reductive tricarboxylic acid cycle; CO2-fixing enzyme
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 CO2are 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 synthase, phosphoenolpyruvate carboxylase,
2-oxoglutarate synthase, and isocitrate dehydrogenase
(IDH) As IDH 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 aerobic bacteria [5], facultative 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 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 a4b4heterooctamers 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)
Trang 2bacteria 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 80 kDa have been
identified from Azotobacter vinelandii [16], Vibrio
parahaemolyticus [17], Rhodomicrobium vannielii [18],
Desulfobacter vibrioformis [19], and Corynebacterium
glutamicum [20] Psychrophilic Vibrio sp strain ABE-1
possesses structurally distinct IDH isozymes of
homodi-meric (IDH-I) and monohomodi-meric (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]
We have previously isolated the green sulfur bacterium
Chlorobium limicolastrain 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°C as described previously [24]
E coliDH5a 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°C When necessary, 50 lgÆmL)1ampicillin
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¢) was a 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 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 programALIGNcontained within the
CLUSTALWprogram provided by DNA Data Bank of Japan (DDBJ) The sequence data was analyzed usingGENETYX
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 ofC limicola idh gene and purification
of the recombinant enzyme
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 idhgene 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)1ampicillin at 37°C Expression of idh in the recombinant cells under the control of T7 promoter was induced for 3 h at 37°C after the addition of 0.1 mM
isopropyl thio-b-D-galactoside (IPTG) when D660 ¼ 0.4 The cells harvested from a 3-L culture were washed twice with 0.1M 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 (0–0.5M) 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
Trang 3Biotech) at a flow rate of 0.35 mLÆmin)1 All purification
steps were carried out at 4°C The active fractions were
examined for apparent homogeneity by SDS/PAGE
Pro-tein concentration was determined by a Bio-Rad ProPro-tein
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 spectrophotometrically at 25°C In the
decarb-oxylic reaction, the assay mixture contained 0.4 mM
triso-diumDL-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, 0.5M stock solution of
NaHCO3in 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°C for 5 min was carried out for
equili-bration before addition of the 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 bufbuf-fers 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
R E S U L T S
Isolation of theidh gene from C limicola
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
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
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
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
Kinetic properties, pH profiles ofCl-IDH and comparison with NADP-dependent IDH fromS cerevisiae
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 Kmvalues for isocitrate and NADP+at the optimum pH were determined to be 45 ± 13 lM and
27 ± 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 CO2concentration 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
Trang 4for the carboxylation reaction Apparent Km values for
2-oxoglutarate and CO2 were 1.1 ± 0.5 mM and
1.3 ± 0.3 mM, respectively, which were much greater than
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 Kmvalues for isocitrate, NADP+, 2-oxoglutarate, and CO2were 20 ± 5 lM, 33 ± 6 lM, 0.85 ± 0.30 mM, and 8.2 ± 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 higher activity for decarboxylation of Sc-IDH (41.0 UÆmg)1) than that for carboxylation (8.7 UÆmg)1) at
pH 7.0 (Fig 2B)
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).
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).
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 K m (l M )
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
V max (UÆmg)1) 38 ± 9 16 ± 2 –
Table 1 Purification of Cl-IDH from recombinant E coli IDH activity was measured with carboxylation reaction.
Step
Total protein (mg)
Total activity (U)
Specific activity (UÆmg)1)
Yield (%)
Purification (fold)
Trang 5Inhibition of carboxylation activity ofCl-IDH
by oxaloacetate
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 mMoxaloacetate (Fig 3A) In contrast, up
to 5 mMoxaloacetate 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 Kivalue of oxaloacetate for
Cl-IDH was determined to be 0.35 ± 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)
D I S C U S S I O N
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
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
Among the intermediates of RTCA cycle, oxaloacetate affected activities of Cl-IDH More than half of the activity was inhibited by 1 mMoxaloacetate in both directions, 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 from
Cr glutamicumand 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
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)1activity in the direction of malate synthesis could be detected (data not shown) However, although 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 thiosulfatophilumgrown in a medium containing3H2O, 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
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 m M (d), 5 m M
(m), and 10 m M (s) 2-oxoglutarate.
Trang 6ATP-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
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