The conformation of the TmIDH structure was open and showed a domain rotation of 25– 30 compared with closed IDHs.. B Overlay of the large domains of TmIDH green, HcIDH blue and PcIDH pi
Trang 1isocitrate dehydrogenase from Thermotoga maritima
Mikael Karlstro¨m1, Ida H Steen2, Dominique Madern3, Anita-Elin Fedo¨y2, Nils-Ka˚re Birkeland2 and Rudolf Ladenstein1
1 Center for Structural Biochemistry, Karolinska Institutet, NOVUM, Huddinge, Sweden
2 Department of Biology, University of Bergen, Norway
3 Institut de Biologie Structurale CEA-CNRS-UJF, Grenoble, France
The enzymes of hyperthermophilic organisms are
remarkably stable and can resist denaturation at
tem-peratures ranging from 80C to above 130 C [1,2],
whereas their counterparts from mesophilic organisms
are usually denatured at around 50C However, they
are often homologous and their catalytic mechanisms
are usually identical [3] Moreover, the gain in free
sta-bilization energy in hyperthermostable proteins,
com-pared with their mesophilic homologues, is generally
small, especially at the growth temperature of the organisms [4] Typically, the net free energy of stabil-ization in both mesophilic and hyperthermophilic pro-teins, is 5–20 kcalÆmol)1, which is equivalent to only
a small number of weak intermolecular interactions [3,5,6] There is no single mechanism or structural fea-ture that is responsible for the high thermotolerance of hyperthermostable proteins [4,7] The most common determinants of hyperthermostability that have been
Keywords
ionic networks; isocitrate dehydrogenase;
thermostability; Thermotoga maritima
Correspondence
M Karlstro¨m, Karolinska Institutet, NOVUM,
Centre for Structural Biochemistry,
S-141 57 Huddinge, Sweden
Fax: +46 8 608 9290
Tel: +46 8 608 9178
E-mail: mikael.karlstrom@biosci.ki.se
(Received 28 February 2006, revised
26 April 2006, accepted 2 May 2006)
doi:10.1111/j.1742-4658.2006.05298.x
Isocitrate dehydrogenase (IDH) from the hyperthermophile Thermotoga maritima (TmIDH) catalyses NADP+- and metal-dependent oxidative decarboxylation of isocitrate to a-ketoglutarate It belongs to the b-decarb-oxylating dehydrogenase family and is the only hyperthermostable IDH identified within subfamily II Furthermore, it is the only IDH that has been characterized as both dimeric and tetrameric in solution We solved the crystal structure of the dimeric apo form of TmIDH at 2.2 A˚ The R-factor of the refined model was 18.5% (Rfree22.4%) The conformation
of the TmIDH structure was open and showed a domain rotation of 25– 30 compared with closed IDHs The separate domains were found to be homologous to those of the mesophilic mammalian IDHs of subfamily II and were subjected to a comparative analysis in order to find differences that could explain the large difference in thermostability Mutational stud-ies revealed that stabilization of the N- and C-termini via long-range elec-trostatic interactions were important for the higher thermostability of TmIDH Moreover, the number of intra- and intersubunit ion pairs was higher and the ionic networks were larger compared with the mesophilic IDHs Other factors likely to confer higher stability in TmIDH were a less hydrophobic and more charged accessible surface, a more hydrophobic subunit interface, more hydrogen bonds per residue and a few loop dele-tions The residues responsible for the binding of isocitrate and NADP+ were found to be highly conserved between TmIDH and the mammalian IDHs and it is likely that the reaction mechanism is the same
Abbreviations
AfIDH, Archaeoglobus fulgidus IDH; ApIDH, Aeropyrum pernix IDH; AUC, analytical ultracentrifugation; BsIDH, Bacillus subtilis IDH; EcIDH, Escherichia coli IDH; HcIDH, human cytosolic IDH; HDH, homoisocitrate dehydrogenase; IDH, isocitrate dehydrogenase; PcIDH, porcine heart mitochondrial IDH; PfIDH, Pyrococcus furiosus IDH; TmIDH, Thermotoga maritima IDH.
Trang 2observed are more ionic interactions at the protein
sur-face and increased formation of large ionic networks
[8–10] Electrostatic optimization and the reduction in
repulsive charge–charge interactions are crucial [11,12]
However, in many cases, the combined effects of
multiple, modestly stabilizing interactions seem to be
responsible for enhanced thermostability Examples
include a reduction in the hydrophobic accessible
sur-face area, increased hydrogen bonding, stronger
inter-subunit interactions, loop deletions and structural
compactness [13–15]
In order to analyse thermotolerance comparatively
within one protein family, we chose isocitrate
dehydrog-enase (IDH), a metal-dependent (Mg2+ or Mn2+)
enzyme in the tricarboxylic acid cycle which catalyses
the subsequent dehydrogenation and decarboxylation
of isocitrate to a-ketoglutarate using NAD+ or
NADP+as a cofactor [16] Owing to its central role in
metabolism, IDH is present in organisms from all three
domains of life, Archaea, Bacteria and Eukarya
Conse-quently, IDH is also present in organisms that have
adapted to a wide range of growth temperatures making
it an attractive model enzyme for studying
heat-adap-tive traits In a previous publication, we characterized
hyperthermostable IDHs from Thermotoga maritima
(TmIDH), Aeropyrum pernix (ApIDH), Pyrococcus
furiosus (PfIDH) and Archaeoglobus fulgidus (AfIDH)
with respect to phylogenetic affiliation, cofactor
specificity, thermostability and oligomeric state, and
identified three different subfamilies of IDH [17]
ApIDH, AfIDH and PfIDH showed high sequence
identity to IDH from the mesophile Escherichia coli
(EcIDH) and they formed, together with all known
archaeal IDHs as well as most bacterial IDHs,
subfamily I Within subfamily II, the bacterial and
eukaryotic NADP+-IDHs were grouped into different
branches However, TmIDH was separated from both
and represented the deepest branch of this subfamily
ApIDH in subfamily I was described as the most
thermostable IDH, with an apparent melting
tempera-ture of 109.9C and TmIDH was described as the
only hyperthermostable IDH known within
sub-family II with an apparent melting temperature of
98.3C Moreover, we identified a heterogeneous
mix-ture of tetrameric and dimeric species of TmIDH in
solution, in which the tetrameric form of TmIDH
repre-sented a unique oligomeric state of NADP-IDH Here,
we report the crystal structure of TmIDH, representing
the first bacterial structure of an IDH from
subfamily II The crystallographic structure is presented
in the dimeric form as crystallization trials of the
tetrameric form of TmIDH have been unsuccessful to
date
In order to reveal possible determinants of the increased thermotolerance of TmIDH, we compared the structure of the dimeric form with the mesophilic mammalian homologues porcine mitochondrial IDH (PcIDH) and human cytosolic IDH (HcIDH) from the same subfamily The observed differences were then compared with differences between ApIDH and its mesophilic homologue EcIDH in subfamily I Further-more, the analysis was used as a guideline to design specific mutants of TmIDH Their properties are dis-cussed with respect to the main mechanisms involved
in protein thermostabilization
Results and Discussion
Purification of the dimeric form of TmIDH
We previously described TmIDH as a heterogenous mixture of dimeric and tetrameric species [17] Recently, the crystal structure of tetrameric homoisoci-trate dehydrogenase from the hyperthermophilic bac-terium Thermus thermophilus was solved and it was suggested that formation of the tetramer was involved
in thermostabilization [18] New analytical ultracentrif-ugation (AUC) data on TmIDH confirmed that two oligomeric species with S20,W values corresponding to
a tetramer ( 7.9 S) and a dimer ( 5.0 S) are present
in the preparation using the previously described puri-fication protocol (data not shown) [17]
In order to separate the two oligomeric forms, a gel-filtration step was added and the separate fractions were analysed using AUC The AUC data demonstra-ted that dimeric and tetrameric species do not re-equilibrate to a heterogenous mixture (Fig 1) Thus, the dimeric and tetrameric forms were not in equilib-rium and could be separated The two forms were con-centrated and subjected to various crystallization trials
To date, crystals of the tetrameric species of TmIDH
0 0,1 0,2 0,3 0,4 0,5 0,6
Sedimentation coefficient (S)
Fig 1 Sedimentation velocity analysis of dimeric Thermotoga
SEDFIT software [62] The single peak is centered on 5,4 S This value corresponds to the one expected for a pure dimer TmIDH.
Trang 3have not been obtained, whereas the dimeric form
pro-duced good quality crystals that were used to solve the
structure
Quality and description of the model
The crystal structure of TmIDH was solved by
molecu-lar replacement at 2.2 A˚ and represents the apo form
of the enzyme The atomic coordinates and structure
factors were deposited in the Protein Data Bank (entry
1ZOR) The model (Fig 2A) was refined to a
crystal-lographic R-value of 18.4% and a free R-value of
22.3% It was crystallized as a dimer in the asymmetric
unit with a solvent content of 54.8% corresponding to
a Matthews’ coefficient of 2.7 A˚3ÆDa)1 The two
sub-units were related by a twofold noncrystallographic
rotation axis and are referred to as subunits A and B
The space group was P212121and the unit cell
parame-ters a¼ 62.5 A˚, b ¼ 88.1 A˚, c ¼ 180.9 A˚ The
sym-metry-related molecules in the crystal did not produce
the tetrameric form Electron-density maps of subunit
A were of very high quality throughout the structure
determination, whereas density maps of the large
domain of subunit B lacked density for many side
chains This was probably caused by a local disorder
in the crystal resulting from the flexibility of the large
domains in combination with the particular crystal
packing However, these side chains were maintained
in the model with ideal conformations (as defined by
the rotamers of the o database) except for residues
B1–3, which were omitted because of dubious main
chain density In subunit A, only three side chains
were lacking density and more solvent molecules were
built compared with subunit B In total, 428 water
molecules were built In both subunits, some residues
were modelled with two conformations with 50%
occupancy each (Lys A48, Lys A62, Glu A81, Lys
A84, Arg A109, Lys A183, Lys A220, Asn A240, Glu
A300, Arg A307, Arg A308, Arg A335, Glu A348, Glu
A
B
C
Fig 2 (A) Ribbon representation of the Thermotoga maritima IDH
(TmIDH) dimer The dimer forms through the association of the
small domains (subunit A: orange and subunit B: light blue) and the
formation of the clasp domain (A: pink and B: purple) Each large
domain (A: green and B: blue) is connected to the small domain via
a flexible hinge region Both subunits were found in an open
con-formation (B) Overlay of the large domains of TmIDH (green),
HcIDH (blue) and PcIDH (pink), showing the N- and C-termini and
the loop deletion of five residues between helices l and m in
TmIDH The domains were superimposed separately because of
different conformations in the subunit (C) Overlay of the small
domains including the clasp domains of TmIDH (green), HcIDH
(blue) and PcIDH (pink) showing the loop deletion of three and four
residues in the clasp domain of TmIDH.
Trang 4B81, Glu B170, Asn B240, Glu B257, Arg B308 and
Glu B385) Four cis-peptides were identified, two of
which are cis-prolines (Pro376 in both subunits) The
rmsd values from ideal geometry were within
reason-able limits and the Ramachandran plot showed that
90.8 and 89.6% of the residues in subunits A and B,
respectively, fall within the most favourable region
Asn159 in subunit B was found in a disallowed
confor-mation, which may be explained by unclear density
and its location in a sharp turn between strand M2
and M3 Table 1 summarizes the quality of the data
and the model
Several crystal structures of different IDHs have
been reported: E coli IDH [19] [EcIDH, PDB codes
3ICD (closed) and 1SJS (open)], Aeropyrum pernix
IDH [20] (ApIDH, PDB codes 1XGV, 1TYO and
1XKD) and Bacillus subtilis IDH [21] (BsIDH, PDB
code 1HQS) representing subfamily I and PcIDH [22]
(PcIDH, PDB code 1LWD) and HcIDH [23] [HcIDH,
PDB code 1T0L (closed) and 1T09 (open)] from
sub-family II They are all homodimeric NADP+-IDHs and their common folds are shared also by the crystal structures of isopropylmalate dehydrogenase which uses a substrate containing the malate moiety in com-mon with isocitrate and belongs to the same family of b-decarboxylating dehydrogenases [24–26] TmIDH showed highest structural similarity to PcIDH and HcIDH, the two other members of subfamily II IDHs for which the structure are known
Hyperthermostable enzymes are often smaller than their homologues from mesophiles The amino acid sequence of TmIDH is shorter than PcIDH and HcIDH TmIDH has 399 amino acid residues, whereas PcIDH and HcIDH contain 413 and 414 residues, respectively However, all of the sequenced bacterial IDHs in this subfamily (i.e also those from mesophiles and psycro-philes) have shorter sequences than the eukaryotic IDHs Therefore, the shorter sequence of TmIDH is most likely a phylogenetic characteristic and not an adaptation to higher temperature All IDH subunits consist of three domains: a large domain, a small domain and a clasp domain (Fig 2A) In TmIDH, resi-dues 1–119 and 281–399 belong to the large domain, res-idues 120–140 and 182–280 constitute the small domain and residues 141–181 form the clasp domain The large domain is connected to the small domain by a flexible hinge region TmIDH was found in an open conforma-tion with relative differences in the rotaconforma-tion of the large domain of 30 compared with the closed ternary iso-citrate–NADP+–Ca2+–HcIDH complex (PDB code: 1T0L) and of 25 compared with the binary iso-citrate–PcIDH complex Compared with the most open subunit of the binary NADP+–HcIDH complex (PDB code: 1T09), TmIDH was 6 more open The clasp domain in TmIDH was typical for subfamily II, with two stacked four-stranded antiparallel b sheets instead
of the two antiparallel a helices beneath a single four-stranded antiparallel b sheet characteristic for sub-family I IDHs
Because of different conformations, the large and the small domains of each homologue were compared separately The rmsd values between the Ca-carbons of the large domain of TmIDH and that of PcIDH and HcIDH were 0.92 A˚ (using 231 Ca-atoms) and 0.97 A˚ (230 Ca-atoms), respectively, when the nonconserved loops in PcIDH and HcIDH were excluded Superposi-tion of the small domains, together with the clasp domain, exhibited rmsd values between TmIDH and PcIDH and HcIDH of 0.84 A˚ (161 Ca-atoms) and 0.88 A˚ (159 Ca-atoms), respectively Overlays of the different domains of TmIDH, HcIDH and PcIDH are shown in Fig 2B,C Because of a transformation of the secondary structure in the open form of HcIDH,
Table 1 Data collection and refinement statistics Values in
paren-theses refer to data in the highest-resolution shell.
TmIDH
(2.24–2.29)
Refinement
(2.236–2.294)
Ramachandran plot (excl Gly and Pro)
rmsd from ideal values
Trang 5which is discussed more in detail below, the separate
domains of the closed form of HcIDH were used in
the superposition
All secondary structural elements were conserved in
TmIDH, PcIDH and HcIDH In total there were 16
a helices (44.9% of the residues in TmIDH) and 16
b strands (19.5% of the residues in TmIDH) In
addi-tion, one 310helix was identified in TmIDH The total
secondary structure content of 65.2% was only slightly
different from that of PcIDH (64.4%) and HcIDH
(66.2%) A structure-based sequence alignment is
shown in Fig 3
TmIDH has a loop deletion of three and four
resi-dues, compared with PcIDH and HcIDH, respectively,
between strands M2 and M3 in the clasp domain
(Fig 2C) Other loop deletions were found between
helices g1 and g2 (one residue), strands E and D (one
residue) and helices l and m (five and four residues,
respectively, Fig 2B) The two latter loop deletions
were also identified in several bacterial IDHs of
meso-philes from this subfamily and must be considered as
phylogenetic characteristics The two first-loop
dele-tions, however, were not found in any mesophilic IDH
This finding is in agreement with earlier observations
that proteins of hyperthermohiles often have shorter
loops [15,27] In this way, fraying elements where the
structure might begin to unfold, are reduced
Differences between the subunits
Subunits A and B of TmIDH were very similar
Super-position of the small domains showed an rmsd of
0.27 A˚ for the Ca-atoms, whereas the rmsd between
the large domains was 0.32 A˚ The relative difference
in the rotation of the large domain between the two
subunits was 2.6
Dimer association
The interface in TmIDH was found to be similar to
that of PcIDH and HcIDH The dimer associates
through the formation of the clasp domain and via
hydrophobic interactions between helices h and i in
both subunits to form a stable four-helix bundle
The active site
The active site of the IDHs is formed in the cleft
between the small and the large domains and contains
residues from both domains and both subunits The
substrate-binding residues of TmIDH were investigated
by superposition of the active site residues from each
domain of TmIDH and the closed substrate-bound
HcIDH and PcIDH separately The putative isocitrate-binding residues from the large domain, Thr77, Ser94 and Asn96, were very well aligned with rmsd values of 0.362 and 0.257 A˚ between TmIDH vs PcIDH and HcIDH, respectively, for all 21 atoms Arg100 and Arg109 are also putative isocitrate-binding residues but showed slightly different conformations in TmIDH, most likely due to the absence of isocitrate in TmIDH However, the isocitrate-binding residues from the small domain, Arg132, Tyr139 and Lys208¢ (the prime indicates the neighbouring subunit of the dimer) were still well aligned with rmsd values of 0.525 and 0.950 A˚ between TmIDH vs PcIDH and HcIDH, respectively, for all 32 atoms The metal-coordinating residues Asp247¢, Asp270 and Asp274 were also struc-turally similar with rmsd values of 0.745 and 1.056 A˚ for all 24 atoms between TmIDH vs PcIDH and HcIDH, respectively (The equivalent of Asp247¢ shows quite a different conformation in HcIDH which
is likely due to the absence of isocitrate in TmIDH.)
In the metal (Mg2+ or Mn2+)-binding site, a Na+ ion was built as it was the only positively charged ion present in the crystallization buffer and no remaining
|Fo|) |Fc| electron density could be observed at 2.0 sigma after it was built The Na+ion was coordinated
by six oxygen ligands in both subunits Two waters (waters 185 and 427 in subunit A and waters 284 and
289 in subunit B), the carbonyl oxygen of Asp270 and Asp247¢ represented the equatorial ligands, whereas Asp270 and Asp274 were the axial ligands
Cofactor binding The b-decarboxylating dehydrogenases share a unique cofactor-binding site that differs from the well-known Rossmann fold found in many other dehydrogenases [19,24,28] Only a few amino acid residues appear to
be responsible for the discrimination between NAD+ and NADP+[29–32] The residues which interact with the 2¢-phosphate of NADP+ are responsible for the discrimination between NAD+⁄ NADP+
All of the residues involved in binding of the NADP+ in the ternary HcIDH complex (PDB code 1T0L) were found to be conserved in TmIDH except a lysine (Lys260 in HcIDH) which interacts with the 2¢-phosphate of NADP+ This lysine was replaced by Arg255¢ in TmIDH and might still be important for the discrimination between NAD+and NADP+ Pre-sumably, the other residues interacting with the 2¢-phosphate in TmIDH are Arg308, His309 and Gln252¢ However, close to Arg308 and His309 there was another arginine (Arg312) in TmIDH directed towards the supposed 2¢-phosphate of NADP+ In
Trang 7PcIDH and HcIDH, this arginine is replaced by a Glu
(318) and Met (318), respectively It is therefore
poss-ible that the cofactor binding is slightly different in
TmIDH compared with PcIDH and HcIDH However,
all other residues putatively involved in binding of
NADP+ were conserved structurally The adenine
ring-binding residues correspond to His303, Val306,
Thr321 and Asn322, whereas Gly304, Thr305 and
Val306 should interact with the 5¢-phosphate of the
adenine Thr77 and Arg82 are equivalent to the
resi-dues which bind the hydroxyl groups of the
nicotina-mide ribose Thr75, Lys72 and Asn96 most likely bind
the amide group of the nicotinamide The rmsd
between TmIDH and the NADP+–isocitrate–HcIDH–
complex (PDB code: 1T0L) was only 0.359 A˚ for all
92 atoms of these residues which do not bind the
2¢-phosphate Between TmIDH and PcIDH without
NADP+, the rmsd was 0.615 A˚ for all 92 atoms
The structural conservation of the isocitrate- and
NADP+-binding residues suggests that isocitrate and
NADP+binding is highly conserved between TmIDH
and the mammalian IDHs and that these enzymes
most likely share the same catalytic mechanism For a
description of the mechanism, see Karlstro¨m et al [20]
and Hurley et al [16]
Putative phosphorylation site
In EcIDH, phosporylation of Ser113 in the so-called
‘phosphorylation loop’ by IDH kinase⁄ phosphatase is
proposed to inactivate the enzyme by blocking the
binding of isocitrate to the active site both sterically
and by electrostatic repulsion of the c–carboxyl group
of isocitrate [33–36] Whether this regulatory
mechan-ism exists in other IDHs is not clear In the closed
ternary complex of HcIDH (PDB code: 1T0L), the
conserved helix i in the small domain is similar to that
observed in all other NADP+–IDH structures and is
part of the four-helix bundle at the dimer interface
However, in the absence of isocitrate, the enzyme has
adopted an open conformation (PDB code: 1T09) and
helix i is found unwound into a loop conformation
where Asp279 interacts with Ser94 of the large domain
in the active site This interaction mimics the
phos-phorylation of the equivalent serine in EcIDH which
inhibits the binding of isocitrate and makes the enzyme inactive It has been postulated that the new interac-tion in HcIDH is competing with isocitrate in binding
to the active site and a self-regulatory mechanism of activity is thereby provided [23] In the open form of TmIDH without isocitrate that we report here, this helix is maintained and the self-regulating mechanism
is therefore not supported The serine in the ‘phos-phorylation loop’ is, however, conserved in TmIDH and PcIDH (Ser94 and Ser95, respectively)
Thermostability
By sequence comparison, PcIDH and HcIDH are 51.3 and 52.2% identical to TmIDH, respectively Neverthe-less, the structural homology makes them suitable for a comparison in order to identify differences which can be related to thermostability The apparent melting tem-perature (Tm) of PcIDH was determined to 59.0C, i.e 39.3C lower than the apparent Tmof TmIDH Below,
we try to relate the large Tm difference to structural determinants that presumably cause this highly increased thermotolerance of TmIDH The revealed dif-ferences between TmIDH and its mesophilic homo-logues in subfamily II are thereafter related to the differences observed between ApIDH and its mesophilic homologue EcIDH in subfamily I The latter compar-ison was made between the open ApIDH (PDB code 1TYO) and the open EcIDH (PDB code 1SJS) and might be slightly different from comparison between ApIDH and the closed EcIDH done previously [20] The sequence identity between TmIDH and ApIDH is only 22.3% (a sequence alignment is shown in Fig 3) The major driving force in protein stability and fold-ing is considered to be ‘the hydrophobic effect’ which results in the burial of most of the hydrophobic resi-dues in the protein core [37,38] The hydrophobic effect explains why many hyperthermostable proteins show a significant increase in the number of buried hydrophobic residues at the core or at subunit interfa-ces, and is sometimes reflected by more hydrophobic residues in the sequence [39–42] This trend could also
be observed in TmIDH which has 42.9% hydrophobic residues, whereas PcIDH and HcIDH have 39.5 and 39.4%, respectively (Table 2) However, many
differ-Fig 3 Structure-based sequence alignment of TmIDH with porcine mitochondrial IDH (PcIDH, PDB code 1LWD), human cytosolic IDH (HcIDH, PDB code 1T0L), Aeropyrum pernix IDH (ApIDH, PDB code 1TYO) and Escherichia coli IDH (EcIDH, PDB code 1SJS) The residues occurring within structurally equivalent regions are boxed Helices and strands appear as cylinders and arrows Conserved residues are shown in green, positions showing conservation of polar or charged character are in bold, those showing conservation of hydrophobic char-acter are in yellow and residues showing a conservation of small size have smaller font Sequence numbering according to TmIDH is red Secondary structure elements were given the nomenclature as implemented in EcIDH [19].
Trang 8Ca
f per
f MM
Trang 9ences between proteins from hyperthermophiles and
mesophiles are observed on the surface of the protein
subunits The contribution of hydrophobic residues at
the protein surface will affect the stability of the
pro-tein unfavorably in aqueous solution, whereas
hydro-philic residues will help to solvate the protein and
thereby stabilize it Accordingly, the accessible surface
area of hyperthermostable proteins is commonly more
polar or charged and less hydrophobic [8,9,11,43–45]
Surface and interface characteristics
First, the open form of TmIDH reported here, was
compared with the open form of HcIDH However,
one must bear in mind that two of the helices (i and i¢)
in the interfacial four-helix bundle are unfolded in the
open HcIDH, giving the surface and the subunit
inter-face a different character Therefore, we show data for
the open HcIDH, the closed HcIDH and the closed
PcIDH in Table 2 It was found that TmIDH has a
6.1% increase in charged accessible surface area, a
1.3% decrease in hydrophobic surface area and 4.8%
decrease in polar surface area compared with the open
HcIDH (Fig 4A and Table 2) This is in line with
ear-lier comparisons between enzymes from
hyperthermo-philes and mesohyperthermo-philes [3,42] In this context, ApIDH is
unusual having a less charged and more polar
access-ible surface than its mesophilic homologue EcIDH
However, the hydrophobic part is still reduced by
0.5% compared with the open EcIDH (Table 2) The
small decrease in the hydrophobic part of the surfaces
might seem insignificant However, using 25 calÆ
mol)1ÆA˚)2for the hydrophobic contribution to the free
energy of folding applied on the difference in accessible
hydrophobic area in TmIDH (which was 2005 A˚2),
gives a free energy difference of 50 kcalÆmol)1
indica-ting a contribution to stability in the expected order of
magnitude [46,47]
Moreover, a reduction of the total solvent-exposed
surface area has also been associated with increased
stability [48,49] The solvent-exposed surface area of
TmIDH was 32 780 A˚2 which is considerably smaller
than the 35 564 A˚2of the open form of HcIDH
How-ever, this is partially due to the shorter sequence of
TmIDH Therefore, the surface-to-volume ratio was
determined [50] For TmIDH it was 0.211, whereas for
the open HcIDH it was 0.224, indicating a slight
decrease in the accessible surface The same trend was
observed between ApIDH (0.208) and EcIDH (0.214)
In principle, a decrease in the accessible surface
might be due to increased burial of the molecular
sur-face at the subunit intersur-face However, both TmIDH
and ApIDH were found to have a smaller relative
interface area compared with their respective homo-logues Because of the unwinding of helix i belonging
to the interfacial four-helix bundle in the open HcIDH,
we preferred to compare the interface area of TmIDH with that of the closed HcIDH and PcIDH It was found that 16.7% of the total molecular surface of the two TmIDH subunits was buried at the interface, whereas 18.5 and 17.6% of the closed HcIDH and PcIDH surfaces were buried, respectively The inter-face of ApIDH was proportionately smaller than both the open and the closed EcIDH (Table 2) [20]
The interface of TmIDH showed a 5% increase in hydrophobic area and a 1.2% decrease in charged area compared with the closed HcIDH (Fig 4B, Table 2)
A
B
Fig 4 (A) Distribution of hydrophobic, polar and charged accessible surface area (ASA) of the different IDHs (B) Distribution of hydropho-bic, polar and charged area at the interface of the different IDHs.
Trang 10A similar trend was found also when the interface of ApIDH was compared with that of the open homo-logue EcIDH However, compared with the closed PcIDH, the interface of TmIDH was 3.5% less hydro-phobic and 3.9% more charged (Table 2) Part of the hydrophobic contribution at the subunit interface in TmIDH involves a cluster of four methionines located
in the interfacial four-helix bundle (Met272 and Met275 from both subunits) This methionine cluster was not present in PcIDH or HcIDH However, below the four-helix bundle, on the top of the clasp domain, there was another, almost identical, intersubunit four-membered methionine cluster (formed by Met176 and Met178 from both subunits), which was found to be conserved in these three IDHs The additional methi-onine cluster in TmIDH might be important for the subunit interaction It was also found that helix h of the interfacial four-helix bundle might be stabilized in TmIDH by three Ala residues in a row instead of one
in PcIDH and two separate Ala residues as in HcIDH Alanine is considered to be the most optimal helix-forming amino acid [51,52]
Aromatic interactions Aromatic interactions are usually identified using a cut-off distance of 7 A˚ between the aromatic ring centres [53] The role of additional aromatic interactions in increasing the thermostability of proteins has been dis-cussed elsewhere [53,54] However, TmIDH was found
to have a decreased fraction of aromatic residues (10.8%) compared with HcIDH (12.1%) and PcIDH (12.7%) However, a cluster of aromatic residues invol-ving Phe205, Phe188, Phe219, Phe223, Tyr215, Tyr241 and His133 was identified in the small domain of TmIDH All of these residues were conserved in PcIDH and HcIDH except Phe205, which has a central posi-tion in this cluster (Fig 5C) A few other nonconserved
A
B
C
Fig 5 (A) The mutation D389N decreased the apparent melting
involved in a nonconserved four-member ionic network with Lys318 and Glu385 (B) Arg186 made interactions with Glu182, Glu225 and Glu226 and was part of a five-member ionic network.
Phe205 was found to have a central position in an aromatic cluster
in the small domain involving Phe188, Phe219, Phe223, Tyr215, Tyr241 and His133 All but Phe205 were conserved within sub-family II The result confirms that aromatic clustering plays a role in increasing the apparent melting temperature of proteins.