Tài liệu Báo cáo khoa học: Crystal structure of thiamindiphosphate-dependent indolepyruvate decarboxylase from Enterobacter cloacae, an enzyme involved in the biosynthesis of the plant hormone indole-3-acetic acid doc

Crystal structure of thiamindiphosphate-dependent indolepyruvate
decarboxylase from
Enterobacter cloacae
, an enzyme involved
in the biosynthesis of the plant hormone indole-3-acetic acid
Anja Schu¨tz
1
, Tatyana Sandalova
2
, Stefano Ricagno
2
, Gerhard Hu¨bner
1
, Stephan Ko¨ nig
1
and Gunter Schneider
2
1
Institute of Biochemistry, Department of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg,
Germany;
2
Division of Molecular Structural Biology, Department of Medical Biochemistry and Biophysics,
Karolinska Institutet, Stockholm, Sweden
The thiamin diphosphate-dependent enzyme indolepyruvate
decarboxylase catalyses the formation of indoleacetaldehyde
from indolepyruvate, one step in the indolepyruvate path-
way of biosynthesis of the plant hormone indole-3-acetic
acid. The crystal structure of this enzyme from Enterobacter
cloacae has been determined at 2.65 A
˚
resolution and refined

to a crystallographic R-factor of 20.5% (R
free
23.6%). The
subunit of indolepyruvate decarboxylase contains three
domains of open a/b topology, which are similar in structure
to that of pyruvate decarboxylase. The tetramer has pseudo
222 symmetry and can be described as a dimer of dimers.
It resembles the tetramer of pyruvate decarboxylase from
Zymomonas mobilis, but with a relative difference of 20° in
the angle between the two dimers. Active site residues are
highly conserved in indolepyruvate/pyruvate decarboxylase,
suggesting that the interactions with the cofactor thiamin
diphosphate and the catalytic mechanisms are very similar.
The substrate binding site in indolepyruvate decarboxylase
contains a large hydrophobic pocket which can accommo-
date the bulky indole moiety of the substrate. In pyruvate
decarboxylases this pocket is smaller in size and allows dis-
crimination of larger vs. smaller substrates. In most pyruvate
decarboxylases, restriction of cavity size is due to replace-
ment of residues at three positions by large, hydrophobic
amino acids such as tyrosine or tryptophan.
Keywords: crystal structure; protein crystallography; pyru-
vate decarboxylase; substrate specificity; thiamin diphos-
phate.
Plant hormones play central roles in the regulation of plant
growth and development. The first plant hormone to be
described was indole-3-acetic acid (IAA), which is synthe-
sized by plants [1,2] and plant-associated bacteria [3,4].
Several pathways for the synthesis of IAA in these
organisms have been described, and most of them start

from
L
-tryptophan as precursor. One of the tryptophan-
dependent biosynthetic routes to IAA is the indolepyruvic
acid (IPA) pathway. This pathway starts from
L
-trypto-
phan, and consists of three steps: (a) the conversion of
tryptophan to indole-3-pyruvic acid; (b) the formation of
indole-3-acetaldehyde; and (c) the production of IAA
(Fig. 1). The first step of the pathway is catalysed by
L
-tryptophan aminotransferase, a pyridoxal-5-phosphate-
dependent enzyme [5]. The intermediate, IPA, is decarboxy-
lated by the action of indolepyruvate decarboxylase (IPDC)
[6] and the resulting indole-3-acetaldehyde is oxidized by
an aldehyde oxidase to IAA [7].
Genes encoding IPDC from several microorganisms
have been cloned and characterized. These organisms
include Enterobacter cloacae [8], Pantoea agglomerans [9],
Klebsiella aerogenes [10], Azospirillum brasilense [11,12] and
Azospirillum lipoferum [13]. The IPDC genes code for
polypeptides of about 550 amino acids in length, corres-
ponding to a molecular mass of  60 kDa per subunit. The
enzyme from E. cloacae, which has been characterized
biochemically to some extent, has a molecular mass of
240 kDa, suggesting a tetrameric structure in solution [6].
The enzyme is dependent on Mg
2+
and thiamin diphos-

phate as cofactors and has a high affinity for the substrate,
indolepyruvate (K
M
¼ 20 l
M
;
1
[13a]). The amino acid
sequences of IPDC show homology to pyruvate decarb-
oxylases (PDC) with, for instance, 40% identity between
IPDC from E. cloacae and PDC from Klyveromyces lactis,
38% identity to PDC from Saccharomyces cerevisiae
(ScPDC) and 32% identity to PDC from Zymomonas
mobilis (ZmPDC) [8].
Correspondence to G. Schneider, Department of Medical Biochemistry
and Biophysics, Tomtebodava
¨
gen 6, Karolinska Institutet,
S-171 77 Stockholm, Sweden.
Fax: +46 8327626, Tel.: +46 87287675,
E-mail:
Abbreviations: IPDC, indolepyruvate decarboxylase; PDC, pyruvate
decarboxylase; EcIPDC, indole-pyruvate decarboxylase from Ente-
robacter cloacae; ZmPDC, PDC from Zymomonas mobilis; ScPDC,
PDC from Saccharomyces cerevisiae;IAA,indole-3-aceticacid;
IPA, indolepyruvic acid; ThDP, thiamin diphosphate.
Note: To facilitate comparison, we are using the nomenclature defined
by Muller et al. (1993) [48] to identify the various domains
in ThDP-dependent enzymes.
(Received 19 January 2003, revised 28 March 2003,

accepted 2 April 2003)
Eur. J. Biochem. 270, 2312–2321 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03601.x
This study reports the three-dimensional structure of
IPDC from E. cloacae, determined to 2.65 A
˚
resolution by
protein crystallography. The fold of the subunit is similar to
that of ScPDC [14] and ZmPDC [15]. However, the packing
of the two dimers in the tetramer is different from that of the
PDCs of known structure, best described as a 20° rotation
of one dimer towards the other when compared to the
Z. mobilis enzyme. The active site shows a substantially
larger substrate binding pocket in IPDC in order to
accommodate the bulky indole moiety of the substrate.
Materials and methods
Protein production and purification
The Escherichia coli strain JM109 harbouring the plasmid
pIP362 (kindly provided by J. Koga, Meiji Seika Kaisha
Ltd, Japan) was used for expression. The plasmid contains
theIPDCgenefromE. cloacae inserted into the high
production vector pUC19. A 6-L culture of Escherichia coli
strain JM109 was grown in a medium containing 2% (w/v)
bactotryptone, 1% (w/v) yeast extract, 0.5% (w/v) sodium
chloride, 0.1 m
M
thiamine, 0.1 m
M
magnesium sulphate,
0.01% (w/v) ampicillin, and 0.15
M

potassium phosphate
pH 6.5 for 24 h at 30 °C. Expression of the IPDC gene was
induced by addition of 1 m
M
isopropyl thio-b-
D
-galacto-
side. Cells were harvested by centrifugation, quickly frozen
in liquid nitrogen and stored at )80 °C. About 25 g of cells
were suspended in 40 mL 0.1
M
potassium phosphate
pH 6.5, containing 10 m
M
thiamin diphosphate (ThDP),
10 m
M
magnesium sulphate, 1 m
M
EDTA, 5 m
M
dithio-
threitol
2
, and disrupted in a French Press at 1200 bar
(Gaulin, APV Homogeniser GmbH, Lu
¨
beck, Germany).
The mixture was centrifuged at 70 000 g for 10 min and the
pellet was discarded. Nucleic acids were precipitated by

incubation with 0.1% (w/v) streptomycin sulphate for
45 min at 8 °C. A 15–30% (w/v) ammonium sulphate
fractionation was performed at a protein concentration of
20 mgÆmL
)1
. After centrifugation at 30 000 g for 5 min, the
precipitate was dissolved in 10 mL 50 m
M
Mes/NaOH
pH 6.5, containing 10 m
M
magnesium sulphate, 0.15
M
ammonium sulphate and 1 m
M
dithiothreitol. The solution
was applied to a Sephacryl S200 H column (5 · 95 cm;
Amersham Biosciences) and eluted with the same buffer at
1mLÆmin
)1
. The IPDC-containing fractions were pooled
and concentrated by precipitation with ammonium sulphate
(0.5 mgÆmL
)1
). After centrifugation the precipitate was
dissolved in 20 m
M
Mes/NaOH pH 6.5, 1 m
M
dithiothrei-

tol and this solution was desalted on a Hiprep column
(2.6 · 10 cm; Amersham Biosciences) and applied to a
Source 15Q column (2.6 · 7 cm; Amersham Biosciences).
Elution was performed using a linear gradient of 120 mL
0–25% of 20 m
M
Mes/NaOH pH 6.5, 1 m
M
dithiothreitol,
0.25
M
ammonium sulphate. The fractions with the highest
catalytic activity and homogeneity were pooled, and after
addition of 0.2
M
ammonium sulphate quickly frozen in
liquidnitrogen,andstoredat)80 °C.
Crystallization
The purified enzyme was concentrated to  4mgÆmL
)1
.
Simultaneously, the buffer was changed to 20 m
M
Mes/
NaOH pH 6.5 and 1 m
M
dithiothreitol. IPDC was crystal-
lized by the hanging drop vapour diffusion method. Crystals
were grown at 20 °C using poly(ethylene glycol) 2000
monomethylether as precipitating agent. Drops contained

equal volumes (2 lL) of reservoir solution [0.1
M
sodium
citrate pH 5.0, 8–12% (w/v) poly(ethylene glycol)] and
IPDC (4 mgÆmL
)1
in 20 m
M
Mes/NaOH pH 6.5, 1 m
M
dithiothreitol, 5 m
M
ThDP, 5 m
M
magnesium sulphate).
Before setting the drops, IPDC was incubated with the
cofactors at room temperature for 30 min. The best crystals
were obtained at 9–10% (w/v) poly(ethylene glycol). Within
3–4 days bundles of needles appeared. Streak seeding was
then used to improve the crystal size. After transfer of seeds
to fresh drops, single crystals appeared within 1 day and
grew to a maximum size of 0.6 · 0.4 · 0.2 mm in 3 days.
Data collection
X-ray data were collected at cryo-conditions with a
ADSC Quantum-4 CCD detector on beam line ID29
(ESRF, Grenoble, France). The crystals were soaked in
crystallization buffer supplemented with 20% glycerol
Fig. 1. The indole-3-pyruvic acid pathway for the biosynthesis of
the plant hormone indole-3-acetic acid in Entero bacter cloa cae. 1,
L

-tryptophan aminotransferase, 2, indolepyruvate decarboxylase, 3,
indoleacetaldehyde oxidase.
Ó FEBS 2003 3D structure of indolepyruvate decarboxylase (Eur. J. Biochem. 270) 2313
before flash freezing directly in the nitrogen stream. The
diffraction data was collected at wavelength 0.979 A
˚
and
processed with
MOSFLM
3
[16]. The CCP4 suite of programs
[17] was used for scaling and reduction of the data. The
space group and cell dimensions were determined using the
auto-indexing option of
MOSFLM
and by the analysis of
pseudo-precession images [18].
Structure determination
The structure was solved by molecular replacement using the
program package
AMORE
[19]. The self-rotation function and
the estimated solvent content [20] indicate that the asym-
metric unit contains four subunits, arranged as a tetramer.
The structure of a dimer of ZmPDC was used as a search
model, as calculation of low resolution models of E. cloacae
PDC (EcPDC) from small angle X-ray solution scattering
data [21] had indicated that the quaternary structure of
IPDC is more similar to that of ZmPDC than ScPDC. A
poly serine model of ZmPDC without cofactors and solvent

atoms was used as search model. The best solution had a
correlation coefficient of 0.235 after rigid body refinement.
This solution was fixed, and the search for the second dimer
gave a solution with a correlation coefficient of 0.32 and an
R-factor of 50.1% after rigid body refinement.
Model building and crystallographic refinement
Refinement of the model was performed with
CNS
4
[22]. To
monitor progress 5% of each data set was set aside for
calculation of R
free
[23]. Initial improvement of the model
was achieved by rigid body refinement, first with the
dimers, and subsequently with the subunits as independent
rigid bodies. As the asymmetric unit contains one
tetramer, tight noncrystallographic symmetry restraints
(Wa ¼ 300 kcalÆmol
)1
ÆA
˚
)2
)
5
were imposed on the crystal-
lographically independent monomers throughout the
refinement procedure. Bulk solvent correction was used
with default CNS parameters. Manual rebuilding of the
model was performed using the program O [24] based on

sigma-weighted 2F
o
) F
c
and F
o
) F
c
electron density
maps. The parts of the polypeptide chain which differ
most from the search model due to insertions/deletions in
the amino acid sequence were modelled based on omit
electron density maps [22].
The coenzyme ThDP was excluded from the search
model and the correctness of the solution was confirmed by
electron density for ThDP and Mg
2+
appearing at the
expected positions (Fig. 2). The model was further refined
by simulated annealing and isotropic B-factor refinement.
Water molecules were modelled using the automatic water
picking option in CNS. All water molecules were checked
for hydrogen bonds with protein atoms. The final R-values
and other refinement statistics are given in Table 1. The
X-ray data and the atomic coordinates have been deposited
at the Protein Data Bank, accession number 1ovm.
Structure analysis
Structure comparisons were carried out using the pro-
grams
TOP

[25] and
O
[24] using default parameters.
Sequence alignments were performed with
MULTALIN
Fig. 2. Stereoview of the ThDP binding site in
IPDC. The initial, unrefined 2Fo-Fc map,
showing the electron density for the bound
magnesium ion and ThDP is contoured at
1 r. The refined protein model is superposed.
The magnesium ion is shown by a green
sphere and red spheres represent bound
solvent molecules.
Table 1. Data collection and refinement statistics.
Space group P2
1
2
1
2
Cell dimensions (A
˚
) 132.2, 151.6, 107.6
Resolution (A
˚
) 2.65
Completeness (%) 99.9 (99.9)
a
Total number of reflections 315 465
Unique reflections 63 426
I/r 7.8 (2.0)

R
sym
(%) 8.7 (36.9)
R (%) 20.5
R
free
(%) 23.6
Number of protein atoms 16404
Number of solvent molecules 347
Root mean square bond lengths 0.007
Root mean square bond angles 1.388
B-factors (A
˚
2
)
Overall 32.2
ThDP 26.0
Solvent 23.4
Ramachandran plot
Percentage of nonglycine residues in:
Favourable regions 87.9
Additionally allowed regions 12.1
a
Numbers in parentheses are for the highest resolution shell.
2314 A. Schu
¨
tz et al. (Eur. J. Biochem. 270) Ó FEBS 2003
( />6
. The analysis of
protein interfaces was done using the Protein–Protein

interaction server ( />server/). Figures were created with
MOLSCRIPT
[26],
BOBSCRIPT
[27] and
RASTER
3
D
[28].
Results
Purification of IPDC
The procedure comprises four steps: streptomycin sulphate
treatment, ammonium sulphate precipitation, gel filtration,
and anion exchange chromatography. The resulting enzyme
is the homogenous apo enzyme, free of cofactors. A
molecular mass of 60 000 Da per subunit resulted from
SDS/PAGE, which corresponded to the value calculated
from the nucleotide sequence of the structural gene. The
identity of the purified enzyme was confirmed by N-terminal
amino acid sequence analysis (Met-Arg-Thr-Pro-Tyr-Cys-
Val-Ala).
Structure determination
The crystals of holo-IPDC belong to the space group P2
1
2
1
2
with unit cell dimensions a ¼ 132.2 A
˚
,b¼ 151.6 A

˚
,c¼
107.6 A
˚
and one tetramer in the asymmetric unit, corres-
ponding to a solvent content of 45%. The structure of IPDC
was solved by molecular replacement using a dimer of
ZmPDC as an initial search model and refined to final R
free
/
R-values of 23.6%/20.5%. The stereochemistry of the
model is as expected for this resolution (Table 1). In
general, the electron density for the polypeptide chain is well
defined. However, there is no continuous electron density
for the long loop connecting the middle and the C-terminal
domains (residues 342–355) and these residues were not
included in the model (Fig. 4,
7
lowercase). The analysis of
the model during refinement showed that almost all residues
obey noncrystallographic symmetry, except the C termini
and the side chains of residues His227, Asp278, Arg367,
Ile379, and Arg394. After superposition of the subunits the
rmsd between all corresponding C
a
atomsis0.13A
˚
for two
monomers in the dimer, and 0.17 A
˚

for two dimers. The
final model includes residues 3–341, and 356–551 of the
protein, four magnesium ions, four molecules of ThDP and
citrate, and 347 water molecules. The crystallographic
refinement statistics are presented in Table 1.
Overall structure of IPDC
IPDC is a homo-tetramer with overall dimensions of
92 · 94 · 116 A
˚
. Each monomer consists of three domains
with an open a/b class topology: the N-terminal PYR
1
domain (residues 3–180), which binds the pyrimidine part of
ThDP; the middle domain (residues 181–340); and the
C-terminal PP domain (residues 356–551), which binds the
diphosphate moiety of the cofactor (Fig. 3). The PYR and
PP domains contain a six-stranded parallel b-sheet flanked
by a number of helices, whereas the middle domain contains
a six-stranded mixed b-sheet (four strands are parallel, two
antiparallel), with several helices packing against the sheet.
The secondary structure elements of IPDC are shown in
Fig. 4, together with the aligned amino acid sequences of
IPDC and ZmPDC. The topology of the IPDC monomer is
similar to that of ScPDC and ZmPDC with some variations
in the length and orientation of helices. The superposition of
the IPDC monomer on the subunit of ScPDC and ZmPDC
results in rmsd of 1.24 A
˚
for 470 out of 563 Ca atoms
and 1.48 A

˚
for 496 out of 568 Ca atoms, respectively. All
insertion/deletions are short, they occur in the loop regions
and do not effect the overall structure. The loop connecting
the middle and PP domain is five residues longer in ZmPDC
and most residues of this loop are invisible in the structure
of IPDC. None of the insertions/deletions occur near the
active site, however, some of them are at the dimerization/
tetramerization interfaces (Fig. 4).
Two monomers interact tightly to form the dimer. The
accessible surface area buried in the monomor–monomer
interface is 3590 A
˚
2
(17% of the whole accessible surface
area). The interface is mostly nonpolar (65% of residues),
but it also contains 26 hydrogen bonds and two salt bridges.
All three domains of the monomer participate in the dimer
interactions (Fig. 4, residues marked ÔdÕ), with most residues
at the interface coming from the PYR and PP domains. This
is in agreement with the average mobility of the domains in
the crystal; the PYR domain has the lowest average B-factor,
23 A
˚
2
(comparable to the B-factor of bound ThDP),
whereas the middle domain has the highest overall B-factor,
37 A
˚
2

. One-hundred and two residues make up the mono-
mer–monomer interface; 57 of these residues are conserved
between IPDC and ZmPDC, and 15 residues are invariant in
all IPDC/PDC sequences (Fig. 4).
The IPDC dimer interface is with 3414 A
˚
2
comparable to
that of pyruvamide-activated ScPDC [29]. In ZmPDC, the
interaction area is larger (4387 A
˚
2
),whereasitissmallerin
Fig. 3. Fold of the subunit of IPDC from Enterobacter cloacae. The
PYR domain is shown in blue, the middle domain in green and the PP
domain in red. The secondary structure elements are labelled as defined
in Fig. 4. The cofactor ThDP and the magnesium ion are included as
ball-and-stick models. The broken line indicates the disordered loop
comprising residues 342–355.
Ó FEBS 2003 3D structure of indolepyruvate decarboxylase (Eur. J. Biochem. 270) 2315
nonactivated ScPDC (2892 A
˚
2
) [14]. The number of
hydrogen bonds is also far fewer than in ZmPDC (26 vs.
66). In part, this is due to the shorter C-terminal region
in IPDC, because the last five residues of ZmPDC are
responsible for a dimer interface area of 400 A
˚
2

. Another
difference of about 400 A
˚
2
in the dimer interface can be
accounted for by a deletion of five residues in the IPDC
amino acid sequence after helix a21 (Fig. 4). In ZmPDC,
residues 496–504, which are inserted at this position,
participate in the monomer–monomer interface. In addition
to these two deletions in the IPDC sequence, there are
several amino acid substitutions resulting in a reduced
number of hydrogen bonds in the IPDC dimer interface, for
instance Ser74 fi Gly, Asn102 fi Gly, Asn104 fi Ala,
Fig. 4. Structural alignment of EcIPDC and ZmPDC sequences. Sequences were denoted as IPDC if biochemical and/or genetic data support such
an activity of the enzyme. Residues conserved in six known/putative IPDCs (Enterobacter cloacae, Pseudomonas putida, Pantoea agglomerans,
Azospirillum brasiliense, Azospirillum lipoferum and Klebsiella aerogenes) are shown in red in the EcIPDC sequence (DCIP_ENTCL). Residues
conserved in 12 PDC sequences (DCP1_MAIZE, DCP1_ORYSA, DCP1_PEA, DCP2_TOBAC, DCP2_ORYSA, DCPY_ZYMMO,
DCP1_YEAST, DCP2_YEAST, DCP3_YEAST, DCPY_KLULA, DCPY_KLUMA, DCPY_HANUV) are also shown in red in the ZmPDC
sequence (DCPY_ZYMMO). Conservative amino acid replacements are shown in blue. Residues common to ZmPDC and IPDC are shown in
bold. a-Helices are displayed as rectangles, b-strands as arrows. ÔdÕ indicates residues in the dimer interface and ÔtÕ residues in tetramer interface.
Residues lining the active site cavity are underlined. Residues binding ThDP are highlighted with a blue background, and those involved in substrate
binding by yellow. Amino acids of EcIPDC invisible in the electron density map are shown in lowercase.
2316 A. Schu
¨
tz et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Gln411 fi Leu, Lys485 fi Ala, Asn486 fi Leu (first resi-
due is that of ZmPDC) (Fig. 4).
Two dimers form a tetramer (Fig. 5), as seen in many
other ThDP-dependent enzymes. However, the dimer–
dimer interface is smaller than the monomer–monomer

interface within the dimer. Only 2030 A
˚
2
(9.5%) of the
dimer accessible surface area is buried in IPDC upon
tetramer formation. That corresponds to 44 interacting
residues, which are marked by ÔtÕ in Fig. 4. Ten of them are
conserved between IPDC and ZmPDC, but none are
invariant in the whole IPDC/PDC family. The majority of
residues contributing to these interfaces is located in
the PYR and middle domains. The interface contains 10
hydrogen bonds in IPDC. The dimer–dimer interface
in IPDC is smaller than the corresponding interface in
ZmPDC (4400 A
˚
2
). It is significantly larger than in non-
activated ScPDC (1344 A
˚
2
), and comparable to pyruv-
amide-activated ScPDC (1920 A
˚
2
)[15].
The tetramer of IPDC differs significantly from other
tetrameric ThDP in the packing of the dimers within the
tetramer. The pseudo 222 symmetry is preserved, and
the molecule can be best described as a Ôdimer of dimersÕ.
The closest relative is ZmPDC, where the second dimer is

rotated by about 20° when tetramers of IPDC and ZmPDC
are compared (Fig. 5). It is noteworthy that the relative
orientation of the dimers in the tetramer is different in all of
the tetrameric PDCs of known three-dimensional structure.
Binding of the cofactors ThDP and Mg
2+
The homo-tetrameric IPDC binds four molecules of the
cofactors ThDP and Mg
2+
. The ThDP binding sites are
located in narrow clefts at the interfaces formed by the PYR
domains from one subunit and the PP domains of the other
subunit within the dimer. ThDP adopts the V-conformation
[30,31] and is completely buried in the cofactor binding cleft.
Several hydrogen bonds that are responsible for binding
and proper orientation of the aminopyrimidine ring, are
conserved in all ThDP-dependent enzymes. One of these,
the hydrogen bond between the N1¢ atom of the pyrimidine
ring of ThDP and the side chain of an invariant glutamate
residue of the neighbouring subunit (Glu52), is essential for
catalysis [32–35]. The C2 carbon atom of the thiazolium ring
points into the active site cavity and is accessible for external
ligands. The diphosphate moiety of ThDP is bound
exclusively to the PP domain of the subunit through
Fig. 5. Quaternary structure of IPDC. Upper panel: stereo view of the quaternary structure of IPDC from Enterobacter cloacae. The four subunits
of the tetramer are shown in different colours. The cofactor molecules are included as ball-and-stick models. Lower panel: different packing of the
dimers in the tetramer of EcIPDC and ZmPDC. After superposition of one dimer in the tetramer of EcIPDC and ZmPDC (green), the difference in
the orientation of the second dimer (IDPC, blue, ZmPDC, red) in the two enzymes is clearly evident.
10
Ó FEBS 2003 3D structure of indolepyruvate decarboxylase (Eur. J. Biochem. 270) 2317

hydrogen bonds and a bridging magnesium ion. The
magnesium ion is octahedrally coordinated to oxygen
atoms from the diphosphate group of ThDP, the side
chains of Asp435 and Asn462, the main chain oxygen atom
of Gly464, and a water molecule. All these interactions are
highly conserved among ThDP-dependent enzymes.
Substrate binding site and catalytic residues
The active site cavity in IPDC extends from the thiazolium
ring of the cofactor to the surface of the protein. The
entrance of the active site cleft is covered by the C-terminal
helix and this part of the polypeptide chain must move in
order to allow entry of the substrate. This structural feature
was also found in ZmPDC [15], and it could be shown that
the kinetic properties of ZmPDC variants, truncated at the
C-terminal helix, are consistent with a role of this helix in
closure of the active site [36].
A model of the a-carbanion/enamine intermediate of the
substrate indole-3-pyruvate with ThDP in the active site of
IPDC was built based on the three-dimensional structure of
the corresponding intermediate in transketolase [37] and the
model derived for ScPDC [38] (Fig. 6). In the immediate
vicinity of the modelled a-carbanion/enamine, there are a
number of invariant amino acids, Asp29, His115, His116,
and Glu468, which are conserved in all PDCs. Site-directed
mutagenesis has confirmed the essentiality of these residues
for catalysis in ScPDC [39,40] and ZmPDC [41–43],
8
respectively. These studies, together with structural data
from crystallography [14,15,29] and modelling [38] have
provided considerable insights into the role of these residues

in PDC. As all amino acids, which were suggested to
participate in catalytic steps of PDC are conserved in IPDC,
the enzymatic mechanism seems to be very similar, if not
identical, in the two enzymes.
A significant difference in the active site between PDC
and IPDC appears to be Gln383, which is replaced by Thr
in most PDCs (Fig. 4). In the structure of holo-IPDC the
side chain of Gln383 points away from the active site cavity
and cannot interact with bound substrate and/or reaction
intermediates. However, only side chain movement would
be sufficient to allow interactions of this residue with bound
substrate, suggesting that Gln383 might be involved in
substrate binding and, possibly, specific recognition of
indole-3-pyruvate.
Substrate recognition
The indole moiety of the modelled intermediate is bound in
a large hydrophobic pocket, lined by residues from three
helices, Ala387, Phe388 (helix a16), Val467, Ile471 (helix
a20), Leu538, Leu542, and Leu546 (a23), and is completely
buried in the protein. Three of these residues (Phe388,
Val467 and Ile471) are either invariant or have conservative
substitutions in all PDC/IPDC sequences (Fig. 4). The
assignment of this hydrophobic pocket as part of the
substrate binding site is further supported by mutational
studies of ZmPDC, because residue substitutions at the
positions corresponding to 467 and 471 in IPDC influence
substrate binding and specificity [43].
The volume of the active site cavity is larger in IPDC
(130 A
˚

3
)thaninZmPDC(85A
˚
3
), where it is partially filled
with bulky amino acids, Tyr290, Trp387, and Trp542
(IPDC sequence numbering). These large aromatic side
chains effectively restrict the size of the pocket and prevent
binding of larger substrates (Fig. 6). The structural model is
thus consistent with the finding that ZmPDC does not
recognize indolepyruvate as a substrate [13a]
9
.Aminoacid
sequence comparisons of residues lining this substrate
recognition pocket reveal identical residues at these posi-
tions also in all plant PDCs. A change in substrate
specificity from pyruvate to indolepyruvate thus involves
at least substitution of three residues in the substrate binding
pocket. In all IPDC sequences, residue 290 is replaced by
threonine, position 387 by alanine or leucine, and position
542 by residues which are smaller than tryptophan, resulting
in a larger cavity size. Restriction of the cavity size thus
seems to be a major cause of discrimination against large
substrates in PDCs.
Yeast PDCs do not follow this substitution pattern as the
basis of discrimination towards large aromatic substrates.
Consequently, ScPDC is, in contrast with ZmPDC, able to
decarboxylate indole-pyruvate (Schu
¨
tz et al. unpublished

data). While yeast PDCs show a similar substitution at
position 290 (Thr fi Phe) as ZmPDC, there are no
replacements at position 387 by amino acids with a large
hydrophobic side chain. Furthermore, there are significant
structural differences between ZmPDC and IPDC on
the one hand, and ScPDC on the other hand involving
the C-terminal part of the polypeptide chain (Fig. 7). In the
latter, differences in the conformation of the loop between
Fig. 6. Stereo picture of the model of the
a-carbanion/enamine intermediate (light grey)
in the active site of EcIPDC. The three resi-
dues, which restrict the size of the substrate
binding cavity in ZmPDC (Tyr290, Trp387
and Trp542), are shown in red.
2318 A. Schu
¨
tz et al. (Eur. J. Biochem. 270) Ó FEBS 2003
strands b11 and b12 prevent the C-terminal helix from
approaching the other subunit in the dimer sufficiently to
shield the active site, as it does in ZmPDC and IPDC. There
is therefore no residue in ScPDC which is structurally
equivalent to 542 in ZmPDC and IPDC. These differences
result in a larger volume of the active site cavity in ScPDC,
allowing accommodation of larger substrates such as
indole-3-pyruvate.
Substrate activation
IPDC follows Michaelis–Menten kinetics (Schu
¨
tz et al.
unpublished data). In this regard, the enzyme is similar to

ZmPDC that in contrast with all other PDCs investigated
so far is not subject to substrate activation [44]. Several
models to account for substrate activation in ScPDC have
been proposed [45–47], involving Cys221 as the site where
the substrate activation cascade is triggered. More recently,
an additional pathway for signal transduction between
active sites in ScPDC has been suggested, based on a
detailed kinetic study [40]. An alternative model is based on
the structure of ScPDC with bound activator pyruvamide,
which revealed a disorder–order transition of two active
site loops (residues 104–113 and 290–304), and which
appears to be a key event in the activation process [29].
These conformational transitions are accompanied by
large-scalechangesintherelativeorientationofthedimers
in the tetramer. In the three-dimensional structure of
ZmPDC,theactivesiteloopsarewellorderedand
observed in a conformation suitable for catalysis to occur
[15]. The much tighter packing of the subunits in the
ZmPDC tetramer, leading to more extensive interactions in
the dimer–dimer interface compared to ScPDC most likely
excludes such large-scale conformational changes during
catalysis, and these structural features explain the lack of
substrateactivationinZmPDC.InIPDC,theassemblyof
the subunits in the tetramer resembles that of ZmPDC
rather than ScPDC. As in ZmPDC, the active site loops
are folded in a conformation poised for catalysis even in
the absence of substrate or other activators. The structure
of IPDC supports the conclusion that the substrate
activation observed in most PDC species may be linked
to the packing of the subunits in the tetramer. Enzyme

species with a rather loose packing such as ScPDC
maintain the possibility of conformational changes during
catalysis, and thus allow for cooperativity, whereas in
ZmPDC and IPDC with tighter and more extensive dimer–
dimer interfaces large-scale conformational changes would
be energetically too costly and are not used for control of
enzyme activity.
Acknowledgements
We thank J. Koga for providing a plasmid producing Enterobacter
cloacae indolepyruvate decarboxylase, and K P. Ru
¨
cknagel (Max-
Planck-Society, Research Unit ÔEnzymology of protein foldingÕ,Halle/
Saale, Germany) for the amino acid sequence analysis. We acknow-
ledge access to synchrotron radiation at beamline ID29, ESRF,
Grenoble. A.S. acknowledges travel support by the Deutscher
Akademischer Austauschdienst (DAAD). This work was supported
by the Science Research Council, Sweden and the Graduiertenkolleg of
Sachsen-Anhalt.
References
1. Bartel, B. (1997) Auxin biosynthesis. Ann. Rev. Plant Physiol.
Plant Mol. Biol. 48, 51–66.
2. Normanly, J. & Bartel, B. (1999) Redundancy as a way of life –
IAA metabolism. Curr. Opin. Plant Biol. 2, 207–213.
3. Kawaguchi, M. & Syono, K. (1996) The excessive production of
indole-3-acetic acid and its significance in studies of the bio-
synthesis of this regulator of plant growth and development. Plant
Cell Physiol. 37, 1043–1048.
4. Patten, C.L. & Glick, B.R. (1996) Bacterial biosynthesis of indole-
3-acetic acid. Can. J. Microbiol. 42, 207–220.

5. Koga, J., Syono, K., Ichikawa, T. & Adachi, T. (1994) Involve-
ment of 1-tryptophan aminotransferase in indole-3-acetic acid
biosynthesis of Enterobacter cloacae. Biochim. Biophsy. Acta 1209,
241–247.
6. Koga, J., Adachi, T. & Hidaka, H. (1992) Purification and char-
acterization of indolepyruvate decarboxylase. J. Biol. Chem. 267,
15823–15828.
7. Sekimoto, H., Seo, M., Kawakami, N., Komano, T., Desloire, S.,
Liotenberg, S., Marion-Poll, A., Caboche, M., Kamiya, Y. &
Koshiba, T. (1998) Molecular cloning and characterization of
aldehyde oxidases in Arabidopsis thaliana. Plant Cell Physiol. 39,
433–442.
8. Koga, J., Adachi, T. & Hidaka, H. (1991) Molecular cloning of the
gene for indolepyruvate decarboxylase from Enterobacter cloacae.
Mol. Gen. Genet. 226, 10–16.
9. Brandl, M. & Lindow, S.E. (1996) Cloning and characterization
of a locus encoding anindolepyruvate decarboxylase involved in
Fig. 7. Comparison of the substrate binding
pockets in IPDC (red) and activated ScPDC
(green). In the latter, the loop between strands
b11 and b12 (shown in blue) prevents the
C-terminal helix a23 from approaching the
active site, and thus leads to a more open
substrate binding pocket in ScPDC. A model
of the a-carbanion/enamine intermediate is
included. The blue sphere indicates the posi-
tion of the Mg
2+
ion.
Ó FEBS 2003 3D structure of indolepyruvate decarboxylase (Eur. J. Biochem. 270) 2319

indole-3-acetic acid synthesis in Erwiniaherbicola. Appl. Environ.
Microbiol. 62, 4121–4128.
10. Zimmer, W., Hundeshagen, B. & Niederau, E. (1994) Demon-
stration of the indolepyruvate decarboxylase gene in different
auxin-producing species of the Enterobacteriaceae. Can. J.
Microbiol. 40, 1072–1076.
11. Costacurta, A., Keijers, V. & Vanderleyden, J. (1994) Molecular
cloning and sequence analysis of an Azospirillum brasilense indole-
3-pyruvate decarboxylase gene. Mol. Gen. Genet. 243, 463–472.
12.Carreno-Lopez,R.,Campos-Reales,N.,Elmerich,C.&Baca,
B.E. (2000) Physiological evidence for differently regulated tryp-
tophan-dependent pathways for indole-3-acetic acid synthesis in
Azospirillum brasilense. Mol. Gen. Genet. 264, 521–530.
13. Yagi, K., Chujo, T., Nojiri, H., Omori, T., Nishiyama, M. &
Yamane, H. (2001) Evidence for the presence of DNA-binding
proteins involved in regulation of the gene expression of indole-3-
pyruvic acid decarboxylase, a key enzyme in indole-3-acetic acid
biosynthesis in Azospirillum lipoferum FS. Biosci. Biotechnol.
Biochem. 65, 1265–1269.
13a. Schu
¨
tz, A., Golbik, R., Tittmann, K., Svergun, D.I., Koch,
M.H.J., Hu
¨
bner,G.&Ko
¨
nig, S. (2003) Studies on structure–
function relationships of indolepyruvate decarboxylase from
Enterobacter cloacae, a key enzyme of the indole acetic acid path-
way. Eur. J. Biochem. 270, 2322–2331.

14. Arjunan, P., Umland, T., Dyda, F., Swaminathan, S., Furey, W.,
Sax,M.,Farrenkopf,B.,Gao,Y.,Zhang,D.&Jordan,F.(1996)
Crystal structure of the thiamine diphosphate-dependent enzyme
pyruvate decarboxylase from the yeast Saccharomyces cerevisiae
at 2.3 A
˚
resolution. J. Mol. Biol. 256, 590–600.
15. Dobritzsch, D., Ko
¨
nig, S., Schneider, G. & Lu, G. (1998) High
resolution crystal structure of pyruvate decarboxylase from
Zymomonas mobilis. J. Biol. Chem. 273, 20196–20204.
16. Leslie, A.G.W. (1992) in Joint CCP4 and ESF-EACMB News-
letter on Protein Crystallography, no. 26. Daresbury Laboratory,
Warrington, UK.
17. Collaborative Computational Project, Number, 4. (1994) The
CCP.4 Suite: Programs for Protein Crystallography. Acta Crys-
tallogr. D50, 760–763.
18. Lu, G. (1999) PATTERN: a precession simulation programme for
displaying reciprocal-space diffraction data. J. Appl. Crystallogr.
32, 375–376.
19. Navaza, J. (1994) AMoRe: an automated package for molecular
replacement. Acta Crystallogr. A50, 157–163.
20. Matthews, B.W. (1968) Solvent content of protein crystals. J. Mol.
Biol. 33, 491–497.
21. Ko
¨
nig, S., Schu
¨
tz, A., Svergun, D.I. & Koch, M.H.J. (2000) First

SAXS measurements on indolepyruvate decarboxylase. Hasylab
Annual Report 295–296.
22. Bru
¨
nger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros,
P., Grosse-Kunstleve, R.W., Jiang, J S., Kuszewski, J., Nilges,
M., Pannu, N.S., Read, R.J., Rice, L.M., Simonson, T. & Warren,
G.L. (1998) Crystallography & NMR system: a new software suite
for macromolecular structure determination. Acta Crystallogr.
D54, 905–921.
23. Brunger, A.T. (1993) Assessment of phase accuracy by cross vali-
dation: the free R value. Methods and applications. Acta Crys-
tallogr. D49, 24–36.
24. Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjeldgaard, M. (1991)
Improved methods for building protein models in electron density
maps and the location of errors in these models. Acta Crystallogr.
A47, 110–119.
25. Lu, G. (2000) TOP: a new method for protein structure compari-
sons and similarity searches. J. Appl. Crystallogr. 33, 176–183.
26. Kraulis, P. (1991) Molscript: a program to produce both detailed
and schematic plots of protein structures. J. Appl. Crystallogr. 26,
282–291.
27. Esnouf, R.M. (1997) An extensively modified version of MolScript
that includes generally enhanced colouring capabilities. J. Mol.
Graph. Model 15 (132–134), 112–113.
28. Merritt, E.A. & Bacon, D.J. (1997) Raster3D: Photorealistic
Molecular Graphics. Methods Enzymol. 277, 505–524.
29. Lu, G., Dobritzsch, D., Baumann, S., Schneider, G. & Ko
¨
nig, S.

(2000) The structural basis of substrate activation in yeast pyru-
vate decarboxylase: a crystallographic and kinetic study. Eur. J.
Biochem. 267, 861–868.
30. Schellenberger, A. (1967) Structure and mechanism of action of
the active center of yeast pyruvate decarboxylase. Angew. Chem. 6,
1024–1035.
31. Shin, W., Pletcher, J., Blank, G. & Sax, M. (1977) Ring stacking
interactions between thiamin and planar molecules as seen in
the crystal structure of a thiamin picrolonatedihydrate complex.
J. Am. Chem. Soc. 99, 3491–3499.
32. Wikner, C., Meshalkina, L., Nilsson, U., Nikkola, M., Lindqvist,
Y., Sundstro
¨
m, M. & Schneider, G. (1994) Analysis of an
invariant cofactor–protein interaction in thiamin diphosphate-
dependent enzymes by site-directed mutagenesis. Glutamic acid
418 in transketolase is essential for catalysis. J. Biol. Chem. 269,
32144–32150.
33. Candy, J.M., Koga, J., Nixon, P.F. & Duggleby, R.G. (1996)
The role of residues glutamate-50 and phenylalanine-496 in
Zymomonas mobilis pyruvate decarboxylase. Biochem. J. 315,
745–751.
34. Killenberg-Jabs, M., Ko
¨
nig, S., Eberhardt, I., Hohmann, S. &
Hu
¨
bner, G. (1997) Role of Glu51 for cofactor binding and cata-
lytic activity in pyruvate decarboxylase from yeast studied by site-
directed mutagenesis. Biochemistry 36, 1900–1905.

35. Kern, D., Kern, G., Neef, H., Tittmann, K., Killenberg-Jabs, M.,
Schneider, G. & Hu
¨
bner, G. (1997) How thiamine diphosphate is
activated in enzymes. Science 275, 67–70.
36. Chang, A.K., Nixon, P.F. & Duggleby, R.G. (2000) Effects of
deletions at the carboxyl terminus of Zymomonas mobilis pyruvate
decarboxylase on the kinetic properties and substrate specificity.
Biochemistry 39, 9430–9437.
37. Fiedler, E., Thorell, S., Sandalova, T., Golbik, R., Ko
¨
nig, S. &
Schneider, G. (2002) Snapshot of a key intermediate in enzymatic
thiamin catalysis: Crystal structure of the a-carbanion of
(a,b-dihydroxyethyl) -thiamin diphosphate in the active site of
transketolase from Saccharomyces cerevisiae. Proc.NatlAcad.Sci.
USA 99, 591–595.
38. Lobell, M. & Crout, D.H.G. (1996) Pyruvate decarboxylase: a
molecular modeling study of pyruvate decarboxylation and acy-
loin formation. J. Am. Chem. Soc. 118, 1867–1873.
39.Liu,M.,Sergienko,E.A.,Guo,F.,Wang,J.,Tittmann,K.,
Hu
¨
bner, G., Furey, W. & Jordan, F. (2001) Catalytic acid-base
groups in yeast pyruvate decarboxylase. 1. Site-directed muta-
genesis and steady-state kinetic studies on the enzyme with the
D28A, H114F, H115F, and E477Q substitutions. Biochemistry 40,
7355–7368.
40. Sergienko, E.A. & Jordan, F. (2001) Catalytic acid-base groups in
yeast pyruvate decarboxylase.3. a steady state kinetic model con-

sistent with the behavior of both wild-type and variant enzymes at
all relevant pH values. Biochemistry 40, 7382–7403.
41. Schenk, G., Leeper, F.J., England, R., Nixon, P.F. & Duggleby,
R. (1997) The role of His113 and His114 in pyruvate decarboxy-
lase from Zymomonas mobilis. Eur. J. Biochem. 248, 63–71.
42. Chang, A.K., Nixon, P.F. & Duggleby, R.G. (1999) Aspartate-27
and glutamate-473 are involved in catalysis of Zymomonas mobilis
pyruvate decarboxylase. Biochem. J. 339, 255–260.
43. Pohl,M.,Siegert,P.,Mesch,K.,Bruhn,H.&Grotzinger,J.(1998)
Active site mutants of pyruvate decarboxylase from Zymomonas
mobilis—a site-directed mutagenesis study of L112, I472, I476,
E473, and N482. Eur. J. Biochem. 257, 538–546.
2320 A. Schu
¨
tz et al. (Eur. J. Biochem. 270) Ó FEBS 2003
44. Bringer-Meyer, S., Schimz, K.L. & Sahm, H. (1986) Pyruvate
decarboxylase from Zymomonas mobilis. Isolation and partial
characterization. Arch. Microbiol. 146, 105–110.
45. Hu
¨
bner, G., Ko
¨
nig, S. & Schellenberger, A. (1988) The functional
role of thiol groups of pyruvate decarboxylase from brewer’s
yeast. Biomed Biochim. Acta 47, 9–18.
46. Baburina, I., Gao, Y., Hu, Z., Jordan, F., Hohmann, S. & Furey,
W. (1994) Substrate activation of brewers’ yeast pyruvate
decarboxylase is abolished by mutation of cysteine 221 to serine.
Biochemistry 33, 5630–5635.
47. Baburina,I.,Li,H.,Bennion,B.,Furey,W.&Jordan,F.(1998)

Interdomain information transfer during substrate activation of
yeast pyruvate decarboxylase: the interaction between cysteine 221
and histidine 92. Biochemistry 37, 1235–1244.
48. Muller, Y.A., Lindqvist, Y., Furey, W., Schulz, G.E., Jordan, F. &
Schneider, G. (1993) A thiamin diphosphate binding fold revealed
by comparision of the crystal structures of transketolase, pyruvate
oxidase and pyruvate decarboxylase. Structure 1, 95–103.
Ó FEBS 2003 3D structure of indolepyruvate decarboxylase (Eur. J. Biochem. 270) 2321