JOURNAL OF BACTERIOLOGY, Mar. 2008, p. 2050–2055
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 190, No. 6
Nondecarboxylating and Decarboxylating Isocitrate Dehydrogenases:
Oxalosuccinate Reductase as an Ancestral Form of
Miho Aoshima* and Yasuo Igarashi
Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo,
Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8567, Japan
Received 14 November 2007/Accepted 3 January 2008
Isocitrate dehydrogenase (ICDH) from Hydrogenobacter thermophilus catalyzes the reduction of oxalosucci-
nate, which corresponds to the second step of the reductive carboxylation of 2-oxoglutarate in the reductive
tricarboxylic acid cycle. In this study, the oxidation reaction catalyzed by H. thermophilus ICDH was kinetically
analyzed. As a result, a rapid equilibrium random-order mechanism was suggested. The affinities of both
substrates (isocitrate and NAD?) toward the enzyme were extremely low compared to other known ICDHs. The
binding activities of isocitrate and NAD?were not independent; rather, the binding of one substrate consid-
erably promoted the binding of the other. A product inhibition assay demonstrated that NADH is a potent
inhibitor, although 2-oxoglutarate did not exhibit an inhibitory effect. Further chromatographic analysis
demonstrated that oxalosuccinate, rather than 2-oxoglutarate, is the reaction product. Thus, it was shown that
H. thermophilus ICDH is a nondecarboxylating ICDH that catalyzes the conversion between isocitrate and
oxalosuccinate by oxidation and reduction. This nondecarboxylating ICDH is distinct from well-known decar-
boxylating ICDHs and should be categorized as a new enzyme. Oxalosuccinate-reducing enzyme may be the
ancestral form of ICDH, which evolved to the extant isocitrate oxidative decarboxylating enzyme by acquiring
higher substrate affinities.
Isocitrate dehydrogenase (ICDH) (EC 126.96.36.199 and EC
188.8.131.52) is an enzyme that catalyzes the oxidative decarboxyl-
ation of isocitrate to 2-oxoglutarate. In prokaryotes, two types
of phylogenetically unrelated ICDHs are known: monomeric
and oligomeric. Prokaryotic oligomeric ICDH is evolutionarily
related to isopropylmalate dehydrogenase (EC 184.108.40.206), tar-
trate dehydrogenase (EC 220.127.116.11), and homoisocitrate de-
hydrogenase (EC 18.104.22.168 and EC 22.214.171.1246); these enzymes
constitute a group called NAD(P)-dependent ?-hydroxyacid
oxidative decarboxylases or NAD(P)-dependent ?-decarbox-
ylating dehydrogenases. The three-dimensional structures of
Escherichia coli ICDH (EcICDH), isopropylmalate dehydro-
genase from Thermus thermophilus, and homoisocitrate dehy-
drogenase from T. thermophilus demonstrate that these en-
zymes share a common fold (11, 12, 17). Prokaryotic ICDH in
this group has previously been considered a homodimeric and
an NAD(P)-dependent enzyme. However, due to increasing
reports of NAD-dependent ICDH in bacteria (Acidithiobacil-
lus, Aquifex, Hydrogenobacter, Methylophilus, and Streptococ-
cus) and archaea (Pyrococcus) and of homotetrameric ICDH
in bacteria (Methylococcus and Thermotoga) (3, 6, 7, 9, 13–15,
22–23), prokaryotic oligomeric ICDH is now recognized as an
enzyme with various oligomeric states and coenzyme specific-
Phylogenetic analyses of prokaryotic oligomeric ICDH
indicate that this enzyme does not comprise a single lineage
but can be divided into many subfamilies (21–23). EcICDH is
one of the best analyzed forms and belongs to a distinctive
subfamily that also contains ICDH from archaea (Aeropyrum,
Archaeoglobus, Caldococcus, and Pyrococcus) and Aquificales
(Aquifex and Hydrogenobacter) (3–6, 21–23). These enzymes
can be considered a single lineage and can be categorized as
We have previously reported an EcICDH-type enzyme from
an organism belonging to the order Aquificales, Hydrog-
enobacter thermophilus (3). The primary sequence of ICDH
from H. thermophilus (HtICDH) is 45.8% identical to that of
EcICDH, although its enzymatic characteristics are quite dif-
ferent (3). In particular, the physiological function of HtICDH
is distinct from that of EcICDH. While EcICDH is involved in
the tricarboxylic acid (TCA) cycle and catalyzes the oxidative
decarboxylation of isocitrate, HtICDH is involved in the re-
ductive TCA cycle and catalyzes the reduction of oxalosucci-
nate (2) (Fig. 1). Thus, differences in the reaction mechanism
between these two enzymes were of great interest.
In this study, we analyzed the kinetic mechanism of the
oxidation reaction catalyzed by HtICDH. As a result, we
clearly demonstrate here that HtICDH is not a conventional
decarboxylating ICDH but a novel nondecarboxylating ICDH.
Furthermore, we suggest a possible hypothesis concerning the
evolutionary history of the prokaryotic oligomeric ICDH
where the oxalosuccinate-reducing enzyme is the ancestral
form of the decarboxylating ICDHs.
MATERIALS AND METHODS
Enzyme preparation. Recombinant ICDH from H. thermophilus TK-6
(IAM12695) (HtICDH), E. coli K-12 (EcICDH), and Caldococcus noboribetus
* Corresponding author. Mailing address: Department of Biotech-
nology, Graduate School of Agricultural and Life Sciences, The Uni-
versity of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8567, Japan.
Phone: 81 3 5841 5143. Fax: 81 3 5841 5272. E-mail: aomiho@mail
?Published ahead of print on 18 January 2008.
(CnICDH) was expressed in E. coli cells and purified as previously described (3,
4). Protein concentrations were determined using bicinchoninic acid protein
assay reagent (Pierce) with bovine serum albumin as a standard.
Kinetic analyses. The steady-state kinetics measurements were performed
spectrophotometrically in a solution (total volume of 400 ?l) containing 100 mM
HEPES-KOH (pH 7.5), 10 mM MgSO4, 100 mM KCl, DL-isocitrate, NAD?, and
7.9 ?g of HtICDH at 50°C. The reaction was started by the addition of isocitrate,
NAD?, and the enzyme, and the initial velocity was monitored at 340 nm (ε ?
6.2 mM?1cm?1) for 30 s.
Kinetic parameters were obtained by varying the DL-isocitrate concentrations
(0.4, 0.7, 1.0, and 4.0 mM) at fixed concentrations of NAD?(2.0, 3.0, and 4.0
mM) and by varying the NAD?concentrations (0.5, 0.75, 1.0, and 4.0 mM) at
fixed concentrations of DL-isocitrate (2.0, 3.0, and 4.0 mM). Product inhibition
was analyzed by the addition of different amounts of NADH (0 to 0.02 mM) to
the assay mixture while the concentration of NAD?was fixed at 4 mM, and the
DL-isocitrate concentration was varied (0.7, 1.0, 2.0, and 4.0 mM); or the con-
centration of DL-isocitrate was fixed at 4 mM, and the NAD?concentration was
varied (0.7, 1.0, 2.0, and 4.0 mM).
Nonlinear regression was performed using KaleidaGraph software (Synergy)
and the Levenberg-Marquardt algorithm. Km
Michaelis constants for DL-isocitrate and NAD?, Ks
substrate constants for DL-isocitrate and NAD?, and KiNADHand Ki?NADHare
the inhibition constants of NADH toward the enzyme and the binary complex,
Chromatographic analyses. The reductive carboxylating activity was con-
firmed by measuring isocitrate formation using ion chromatography. The reac-
tion was performed in a mixture (total volume of 400 ?l) containing 100 mM
bicine [N,N-bis(2-hydroxyethyl)glycine]-KOH (pH 6.5), 10 mM MgCl2, 20 mM
2-oxoglutarate, 50 mM NaHCO3, 5 mM NADPH, and ICDH. NADH was used
instead of NADPH in the case of HtICDH. The reaction temperatures used were
37°C for EcICDH and 70°C for HtICDH and CnICDH. A total of 7.3 ?g of
EcICDH, 6.2 ?g of CnICDH, and 16 ?g of HtICDH was used. After incubation
for 5 min, the reaction mixture was cooled on ice-cold water, diluted with MilliQ
water (Millipore) if required to yield the optimal chromatogram, and injected
onto the column.
Oxalosuccinate released as a product during the oxidation reaction was also
detected using ion chromatography. The reaction was performed in a mixture
(total volume of 400 ?l) containing 100 mM bicine-KOH (pH 9.5), 10 mM
MgCl2, 2 mM DL-isocitrate, 2 mM NADP?(for EcICDH) or 2 mM NAD?(for
HtICDH), and ICDH. Eighteen micrograms of EcICDH and 79 ?g of HtICDH
were used. After incubation for 20 min at 20°C, the reaction mixture was cooled
on ice-cold water, diluted with MilliQ water if required to yield the optimal
chromatogram, and injected onto the column.
The chromatographic system and the precise chromatographic conditions used
were identical to those described elsewhere (2).
DL-isocitrateand KmNADare the
DL-isocitrateand KsNADare the
Reductive carboxylation of 2-oxoglutarate. We have previ-
ously reported that HtICDH is unable to catalyze the reductive
carboxylation of 2-oxoglutarate (3), whereas in the case of
EcICDH, by contrast, an ability to catalyze the reductive car-
boxylation of 2-oxoglutarate has been shown and well analyzed
(8). The reversibility of EcICDH was evident from the pres-
ence of isocitrate in the chromatogram (Fig. 2A). The ability to
catalyze the reductive carboxylation of 2-oxoglutarate seems to
be a common feature of the EcICDH-type enzymes since isoci-
trate formation was also observed when CnICDH, the enzyme
from a hyperthermophilic archaeon C. noboribetus (4, 5), was
used (Fig. 2B). However, isocitrate formation was not detected
when HtICDH was used (Fig. 2C). Therefore, although
HtICDH shows high sequence similarity with EcICDH and
CnICDH, these results suggest that differences exist between
the reaction mechanisms of these enzymes.
Kinetic analysis. HtICDH utilizes NAD?instead of NADP?,
and the high apparent Kmvalues (for both isocitrate and
NAD?) and the low kcatvalues (3) indicate that it is an inef-
fective decarboxylating enzyme. In order to elucidate the ki-
netic mechanism of HtICDH, further precise measurements
were carried out. Under the conditions of fixed NAD?and
variable isocitrate concentrations, enzymatic activities were
measured by monitoring NAD?reducing activity. Excess
amounts of MgSO4were added to the assay mixtures so that
isocitrate could form a complex with Mg2?ions. The double-
reciprocal plots gave three linear lines with intersections in the
second quadrant (Fig. 3A). A similar profile, with intersections
in the second quadrant, was also observed when fixed isocitrate
and variable NAD?concentrations were used (Fig. 3B). These
observations suggest the possibility of a rapid equilibrium ran-
dom-order or an ordered mechanism and exclude the possibil-
ity of a ping-pong mechanism. Most of the past studies support
a random mechanism for this enzyme irrespective of its origin
FIG. 1. Physiological roles of ICDH in E. coli and in H. thermophi-
lus. (A) EcICDH is an enzyme involved in the TCA cycle and catalyzes
the oxidative decarboxylation of isocitrate to form 2-oxoglutarate.
(B) HtICDH is an enzyme involved in the reductive TCA cycle and
catalyzes oxalosuccinate reduction, which corresponds to the second
step of the reductive carboxylation of 2-oxoglutarate.
FIG. 2. Reductive carboxylation catalyzed by ICDH. Chromato-
grams of the mixtures obtained after the reductive carboxylation
reaction by EcICDH (incubated for 5 min at 37°C using 7.3 ?g of
protein) (A), CnICDH (incubated for 5 min at 70°C using 6.2 ?g of
protein) (B), and HtICDH (incubated for 5 min at 70°C using 16 ?g
of protein) (C). Reactions were performed in a volume of 400 ?l
containing 100 mM bicine-KOH (pH 6.5), 10 mM MgCl2, 20 mM
2-oxoglutarate, 50 mM NaHCO3, 5 mM NADPH (for EcICDH and
CnICDH) or 5 mM NADH (for HtICDH), and ICDH. After incu-
bation, the reaction mixtures were diluted with water (threefold
dilution) before injection onto the column.
VOL. 190, 2008NONDECARBOXYLATING AND DECARBOXYLATING ICDHs2051
(8). If a random mechanism is also true for this enzyme, it is
possible to interpret the profiles obtained as showing noninde-
pendent binding of the two substrates (isocitrate and NAD?).
Although the three intersections vary rather widely (indicated
by a circle in Fig. 3A), Ks
from the average x coordinate value of the intersections. From
the secondary plot of 1/kcat
for each NAD?concentration) against 1/[NAD?], Km
kcatvalues were calculated to be 0.47 mM and 4.3 s?1, respec-
tively. From the secondary plot of the slope against 1/[NAD?],
x coordinate value of the intersections of the second profile
(indicated by a circle in Fig. 3B), Ks
Product inhibition analysis. In order to elucidate further the
precise kinetic mechanism of HtICDH, product inhibition
analysis was carried out. Enzymatic activity in the presence of
different concentrations of NADH was measured by monitor-
ing NAD?reducing activity. When fixed NAD?and variable
isocitrate concentrations were used, the double-reciprocal
plots gave three linear lines with intersections in the second
quadrant (Fig. 3C). When fixed isocitrate and variable NAD?
concentrations were used, the double-reciprocal plots gave in-
tersections on the y axis (Fig. 3D). The profiles obtained
strongly suggest a rapid equilibrium random-order mechanism
DL-isocitratewas calculated to be 4.0 mM
appis an apparent kcatvalue
DL-isocitratewas calculated to be 0.20 mM. From the average
NADwas calculated to be
with abortive complex (isocitrate-enzyme-NADH) formation.
NADH is a noncompetitive (or mixed competitive) inhibitor
against isocitrate, and from the Dixon plot, Ki
values were calculated to be 0.02 mM and 0.13 mM, respec-
tively. NADH is a competitive inhibitor against NAD?, and
from the Dixon plot, a consistent Ki
Another possible product, 2-oxoglutarate, did not exhibit an
inhibitory effect on the enzymatic activity of HtICDH (at least
up to 10 mM). This observation indicates that 2-oxoglutarate
does not compete with isocitrate and suggests that 2-oxogluta-
rate does not bind to HtICDH. Thus, it is highly probable that
2-oxoglutarate is not a product of the enzymatic reaction cat-
alyzed by HtICDH.
Oxalosuccinate production. If 2-oxoglutarate is not a product
of the enzymatic reaction catalyzed by HtICDH, the genuine
product is most likely to be oxalosuccinate. Oxalosuccinate will
be produced if HtICDH catalyzes only dehydrogenation and if
the following decarboxylation is a nonenzymatic reaction. This
is an extraordinary idea since it is well established that, for
EcICDH, the dehydrogenation and decarboxylation reactions
are closely coupled (10). In the case of NADP-dependent
ICDH from pig heart, the hypothetical intermediate, oxalosuc-
cinate, is not released from the enzyme (20). Furthermore,
since enzyme-bound oxalosuccinate is not detectable, it is
NADHvalue (0.02 mM) was
FIG. 3. Kinetics of the oxidation reaction catalyzed by HtICDH. (A) Double-reciprocal plots obtained by varying the isocitrate concentration
at several different fixed concentrations of NAD?(circle, 4 mM; square, 3 mM; triangle, 2 mM). (B) Double-reciprocal plots obtained by varying
the NAD?concentration under several different fixed concentrations of DL-isocitrate (circle, 4 mM; square, 3 mM; triangle, 2 mM). (C) Inhibition
by NADH versus isocitrate. The concentration of NAD?was fixed at 4 mM, and the DL-isocitrate concentration was varied at several different
concentrations of NADH (circle, 0 mM; square, 0.01 mM; triangle, 0.02 mM). (D) Inhibition by NADH versus NAD?. The concentration of
DL-isocitrate was fixed at 4 mM, and NAD?concentration was varied at several different concentrations of NADH (circle, 0 mM; square, 0.01 mM;
triangle, 0.02 mM). v, velocity.
2052AOSHIMA AND IGARASHI J. BACTERIOL.
likely that oxalosuccinate is not a true intermediate of the
ICDH reaction (19–20). Nevertheless, the hypothesis that
HtICDH produces free oxalosuccinate as a reaction product
was attractive since it would provide a definite answer to the
question of why HtICDH cannot catalyze the reductive car-
boxylation of 2-oxoglutarate. In order to confirm the hypoth-
esis, we established a detection system for the formation of
oxalosuccinate. Since oxalosuccinate is very unstable and
readily decarboxylated to yield 2-oxoglutarate, the enzymatic
reaction was performed at a low temperature (20°C). In the
case of EcICDH, oxalosuccinate was not detected (Fig. 4A),
which is consistent with the studies so far reported. By contrast,
in the case of HtICDH, the formation of oxalosuccinate was
clearly observed (Fig. 4B). Since oxalosuccinate was clearly
detectable as a product, HtICDH was shown to be a nonde-
Decarboxylating and nondecarboxylating ICDH. ICDH is
known as an enzyme that catalyzes consecutive dehydrogena-
tion and decarboxylation reactions in a closely coupled man-
ner. However, here we clearly demonstrate that HtICDH is not
a decarboxylating ICDH but a nondecarboxylating ICDH.
HtICDH catalyzes only the oxidation (dehydrogenation) of
isocitrate to oxalosuccinate, and the subsequent decarboxyl-
ation of oxalosuccinate to 2-oxoglutarate is nonenzymatic (Fig.
5). Physiologically, HtICDH catalyzes the reverse of this reac-
tion, i.e., the reduction of oxalosuccinate to isocitrate, and,
thus, it would be more appropriate to call this enzyme oxalo-
succinate reductase. As such, HtICDH catalyzes only the con-
version between isocitrate and oxalosuccinate by oxidation and
reduction (with no decarboxylation or carboxylation), which is
completely different from other known conventional ICDHs.
Since no nondecarboxylating ICDHs have been reported pre-
viously, HtICDH should be assigned to a new category of
enzymes with a new EC number.
We have previously reported that HtICDH is unable to
catalyze the reductive carboxylation of 2-oxoglutarate (3). The
reason for this phenomenon can now be clearly understood
because this reaction is not performed by HtICDH, and 2-oxo-
glutarate is neither a substrate nor a product of HtICDH. This is
supported by the observation that 2-oxoglutarate did not act as
a product inhibitor of HtICDH.
In this study, we demonstrated that two types of ICDH exist:
decarboxylating and nondecarboxylating. This is analogous to
malate dehydrogenase, for which both nondecarboxylating
(EC 126.96.36.199 and EC 188.8.131.52) and decarboxylating (EC
184.108.40.206, EC 220.127.116.11, EC 18.104.22.168, and EC 22.214.171.124) enzymes
are known. Similarly, both decarboxylating and nondecarboxy-
lating forms of tartrate dehydrogenase exist (EC 126.96.36.199 and
EC 188.8.131.52, respectively) (24). Although this is the first report
of a nondecarboxylating ICDH, it is possible that this type of
ICDH is widely distributed but difficult to distinguish. It is
especially difficult to distinguish between the decarboxylating
and nondecarboxylating forms when the oxidation reaction is
spectrophotometrically analyzed. It may be possible to identify
additional nondecarboxylating ICDHs from other organisms
by screening for the inability to catalyze the reductive carbox-
ylation of 2-oxoglutarate or to be inhibited by 2-oxoglutarate,
as these are the distinguishing features of the nondecarboxy-
Kinetic features of HtICDH. The kinetic analyses performed
strongly suggest that the enzymatic reaction catalyzed by
HtICDH, in the direction of oxidation, proceeds by a random
mechanism where either of the substrates, isocitrate or NAD?,
can bind to the enzyme first, as in the case of EcICDH (8). The
proposed kinetic mechanism is outlined in Fig. 6. Interestingly,
the binding of the two substrates (isocitrate and NAD?) is not
independent. In the case of isocitrate, the Michaelis constant
the substrate constant (Ks
indicates that the affinity toward isocitrate is increased by the
binding of NAD?. The reciprocal effect applies in the case of
NAD?. The Michaelis constant (Km
order of magnitude lower than the substrate constant (Ks
5.4 mM), and thus, the binary complex (isocitrate-enzyme)
(Fig. 6, IC-Enz) has a higher affinity for NAD?than the en-
zyme with no substrate bound (Fig. 6, Enz). Although the
Michaelis constants of HtICDH are lower than the substrate
constants, they are both extremely high compared to those of
EcICDH (8). Thus, it can be said that HtICDH is quite inef-
DL-isocitrate, 0.20 mM) is one order of magnitude lower than
DL-isocitrate, 4.0 mM). This observation
NAD, 0.47 mM) is one
FIG. 4. Oxalosuccinate formation by the oxidation reaction. Chro-
matograms of the mixture obtained after the oxidation reaction by
EcICDH (using 18 ?g of protein) (A) and HtICDH (using 79 ?g of
protein) (B). The reaction was performed in a volume of 400 ?l
containing 100 mM bicine-KOH (pH 9.5), 10 mM MgCl2, 2 mM
DL-isocitrate, 2 mM NADP?(for EcICDH) or 2 mM NAD?(for
HtICDH), and ICDH. After incubation at 20°C for 20 min, the reac-
tion mixture was diluted with water (12-fold in A and 2-fold in B)
before injection onto the column.
FIG. 5. Oxidation reaction catalyzed by HtICDH. HtICDH only
catalyzes the oxidation of isocitrate to oxalosuccinate. The following
decarboxylation is a nonenzymatic reaction.
VOL. 190, 2008NONDECARBOXYLATING AND DECARBOXYLATING ICDHs2053
fective as an oxidative enzyme. Taking into account the high
substrate constants, the oxidation reaction becomes possible
only under conditions where the concentration of at least one
of the substrates is extremely high. Because such a high con-
centration of isocitrate or NAD?is unlikely to occur in H.
thermophilus cells under physiological conditions, it is highly
probable that HtICDH cannot function as an oxidative enzyme
in this organism. As such, the kinetic parameters obtained in
this study strongly suggest that HtICDH is not an oxidative
enzyme but a reducing enzyme.
Product inhibition analysis performed during this study re-
vealed that NADH is a potent inhibitor of HtICDH, compet-
ing for the NAD binding site with NAD?. Inhibitor constants
toward the enzyme (Ki
complex (isocitrate-enzyme; Ki?NADH, 0.13 mM) were similar
to the inhibitor constants of NADPH toward EcICDH (8).
Although we have not examined the level of cytosolic NADH
in H. thermophilus, it may have an inhibitory effect on the
oxidation activity of HtICDH.
Since HtICDH is a reducing enzyme in H. thermophilus, the
kinetic mechanism analyzed in this study is the reverse of
the physiological reaction. If the oxidation reaction pathway
analyzed here accurately represents the reverse of the reduc-
tion reaction pathway, it may be possible to make deductions
about the product-releasing mechanism of the reduction reac-
tion from the substrate-binding mechanism of the oxidation
reaction. If this is the case, once one of the products (isocitrate
or NAD?) has been released from the enzyme, the other will
then be released more readily. This mechanism may also assist
in driving the enzymatic reaction in a reductive direction.
Oxalosuccinate reductase as an origin of ICDH. HtICDH
is quite different from other known ICDHs with respect to its
reaction mechanism. HtICDH catalyzes a bi-bi reaction (oxi-
dation and reduction between isocitrate and oxalosuccinate)
while conventional ICDHs catalyze a bi-ter reaction (in which
carbon dioxide is involved). Thus, as we have proposed, it may
be more appropriate to redesignate HtICDH an reductase.
Nevertheless, a close relationship between HtICDH and other
NADH, 0.02 mM) and toward the binary
ICDHs is apparent when the structures of these proteins are
examined. The primary sequence of HtICDH is 45.8% identi-
cal to that of EcICDH, and both sequences show overall sim-
ilarity (3). This observation indicates that both enzymes share
a common fold, and from the phylogenetic viewpoint, they
undoubtedly belong to the same subfamily and have evolved
from a common ancestral enzyme. Since H. thermophilus has
been assigned to the most deeply branching lineage of the
domain Bacteria (18), it may be possible that HtICDH is an
ancestral form of EcICDH. Therefore, we suggest a possible
hypothesis as to how the enzyme evolved from an oxalosucci-
nate-reducing enzyme to an isocitrate oxidative decarboxylat-
In the case of EcICDH, during the enzymatic reaction, isoci-
trate is oxidized to oxalosuccinate and then oxalosuccinate is
decarboxylated to 2-oxoglutarate. Oxalosuccinate may be an
enzyme-bound intermediate, but it is never released from the
enzyme. This suggests that oxalosuccinate binds more tightly
than isocitrate or 2-oxoglutarate. The affinity of isocitrate is
higher than that of 2-oxoglutarate, as shown by their respective
Michaelis constants (8). Accordingly, the affinities toward
EcICDH can be ranked in the order of the highest to the
lowest as oxalosuccinate ? isocitrate ? 2-oxoglutarate, which
corresponds to intermediate ? substrate ? product (Fig. 7).
This would be the preferred order for an isocitrate oxidative
It is highly probable that, irrespective of the origin of ICDH,
the order of the substrate affinities toward the enzyme is always
the same (oxalosuccinate ? isocitrate ? 2-oxoglutarate) and
only the absolute affinities of the three vary. Since oxalosucci-
nate, isocitrate, and 2-oxoglutarate bind to the same site on the
enzyme, the affinities of these three ligands may be closely
interrelated. This interdependence between affinities is a com-
mon feature for multiple ligands that bind to the identical site
on enzymes. For example, an interdependence between the
affinities of NADP and NADPH has been reported for the
engineered isopropylmalate dehydrogenase from E. coli (16).
As shown in Fig. 7, if the affinities of oxalosuccinate, isoci-
trate, and 2-oxoglutarate are lowered simultaneously, the char-
acteristics of the enzyme change significantly. Oxalosuccinate,
although it still binds tightly, can be released from the enzyme.
This means that oxalosuccinate is no longer an enzyme-bound
FIG. 6. Proposed kinetic mechanism of the oxidation reaction cat-
alyzed by HtICDH. Enz, HtICDH; IC, isocitrate; filled circle, isocitrate
binding site of the binary complex (high affinity); open circle, isocitrate
binding site of HtICDH (low affinity); filled star, NAD binding site of
the binary complex (high affinity); open star, NAD binding site of
HtICDH (low affinity). Arrows with dotted lines indicate product in-
hibition by NADH.
FIG. 7. Hypothetical evolutionary pathway from an oxalosuccinate-
reducing enzyme to an isocitrate-oxidative decarboxylating enzyme.
The affinities of oxalosuccinate, isocitrate, and 2-oxoglutarate toward
the enzyme are shown. H. thermophilus ICDH, an oxalosuccinate-
reducing enzyme, is proposed to have evolved to an isocitrate-oxidative
decarboxylating enzyme (E. coli ICDH) by acquiring higher substrate
2054 AOSHIMA AND IGARASHI J. BACTERIOL.
intermediate but, rather, a substrate. Isocitrate becomes a
product because of its low affinity. 2-Oxoglutarate cannot bind
to the enzyme, so it is no longer a substrate or a product. These
properties agree exactly with the characteristics of the oxalo-
succinate-reducing enzyme, HtICDH. Thus, it can be said that
the oxalosuccinate-reducing enzyme corresponds to ICDH
with a lowered substrate affinity. If the phylogenetic history of
ICDH is traced chronologically, it is likely that the oxalosuc-
cinate-reducing enzyme is the ancestral form, and that this
evolved to the isocitrate oxidative decarboxylating enzyme by
acquiring higher substrate affinities. Accordingly, it is highly
probable that oxalosuccinate reductase is the origin of extant
ICDH and that an ancient type of enzyme still remains in H.
thermophilus. Since ancestral forms of the TCA cycle enzymes
are abundant in H. thermophilus (1), it is a crucial organism for
the further investigation of the evolutionary history of the TCA
As described, the substrate affinities of HtICDH and
EcICDH are significantly different. However, residues involved
in the substrate (isocitrate-Mg2?) binding in EcICDH are all
conserved in the HtICDH sequence (3). As yet, we have not
determined which residues in HtICDH are responsible for the
low affinity toward the substrates. A preliminary three-dimen-
sional comparison study using a homology modeling tool sug-
gests that the position of D284 in HtICDH has widely diverged
from that of the corresponding residue (D283) in EcICDH.
Since D283 is one of the substrate binding residues in EcICDH
(10), this divergence may be the cause of the low substrate
affinity of HtICDH. Further molecular dynamics, crystallo-
graphic, and mutagenesis studies are required.
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VOL. 190, 2008NONDECARBOXYLATING AND DECARBOXYLATING ICDHs2055