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

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-

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

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-

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

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

[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)

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

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

. 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

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

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

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

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

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

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

(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.

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)