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 220.127.116.11 and EC
18.104.22.168) 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 22.214.171.124), tar-
trate dehydrogenase (EC 126.96.36.199), and homoisocitrate de-
hydrogenase (EC 188.8.131.52 and EC 184.108.40.2066); 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.
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