Studies on structure–function relationships of indolepyruvate
decarboxylase from
Enterobacter cloacae
, a key enzyme
of the indole acetic acid pathway
Anja Schu¨tz
1
, Ralph Golbik
1
, Kai Tittmann
1
, Dmitri I. Svergun
2,3
, Michel H. J. Koch
2
, Gerhard Hu¨ bner
1
and Stephan Ko¨ nig
1
1
Institut f
€
uur Biochemie, Fachbereich Biochemie/Biotechnologie, Martin-Luther-Universit
€
aat Halle-Wittenberg, Halle, Germany;
2
European Molecular Biology Laboratory, Hamburg Outstation, Hamburg, Germany;
3
Institute of Crystallography,
Russian Academy of Sciences, Moscow, Russia
Enterobacter cloacae, isolated from the rhizosphere of

The auxin indole-3-acetic acid, a phytohormone that
promotes cell growth and elongation and influences rooting,
is produced by plants [1,2] and plant-associated bacteria
[3,4]. Both tryptophan-dependent and -independent path-
ways of indole-3-acetic acid synthesis have been described
[5,6]. Plants use several mechanisms to control levels of the
active auxin indole-3-acetic acid. Thus, during different
developmental stages, indole-3-acetic acid may originate
from diverse sources for different auxin requirements, and
under different environmental conditions. Bacteria primar-
ily use tryptophan-dependent pathways. Phytopathogenic
strains follow the indoleacetamide pathway and plant
growth promoting strains the indolepyruvate pathway
(Fig. 1). Indolepyruvate decarboxylase (IPDC), a key
enzyme in the second pathway, is a thiamine diphosphate
(ThDP)- and Mg
2+
-dependent homotetrameric enzyme
that catalyses the decarboxylation of indole-3-pyruvate to
indole-3-acetaldehyde [7–9]. Several microbial genes enco-
ding IPDC have been reported, including one from
Enterobacter cloacae isolated from the rhizosphere of
actively growing cucumbers [10]. DNA sequence analyses
revealed only one gene encoding EcIPDC. Its predicted
amino acid sequence comprises 552 residues and has
40% identity to PDC from Kluyveromyces lactis (DCPY
KLULA), 38% to PDC from Saccharomyces cerevisiae
(DCP1 YEAST), and % 32% to PDC from Zea mays
(DCP1 MAIZE), Oryza sativa (DCP1 ORYSA), Pisum
sativum (DCP1 PEA), and to PDC from Zymomonas

EC 4.1.1.1).
Note:S
05
is the substrate concentration at half-maximum reaction
rate for enzymes displaying cooperativity characterized by sigmoid
reaction rate vs. substrate concentration plots.
(Received 5 February 2003, revised 17 March 2003,
accepted 2 April 2003)
Eur. J. Biochem. 270, 2322–2331 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03602.x
The pyruvate derivatives a-keto glutarate and b-phenyl-
pyruvate inhibit EcIPDC activity. Indole and some similar
metabolites such as
L
-tryptophan, indole-3-lactate, indole-
3-acetaldehyde, tryptophol, and indole-3-acetate have
no effect on the enzymatic activity at a concentration of
0.5 m
M
[7].
Below, results on fast kinetics, substrate specificity, and
cofactor binding of EcIPDC are presented. For the kinetic
measurements a continuous optical assay was developed.
The pH- and cofactor-dependent subunit association beha-
viour was studied by small angle X-ray solution scattering.
The catalytic specificities of EcIPDC, ScPDC, and ZmPDC
for various substrates are discussed on the basis of their
crystal structures.
Materials and methods
Reagents
Horse liver alcohol dehydrogenase was from Roche

potassium phosphate pH 6.5, containing 10 m
M
ThDP,
Fig. 1. Scheme of the postulated biosynthesis pathway of indole-3-acetate from
L
-tryptophan in E. cloacae including the keto-enol tautomerism of
indolepyruvate, modified according to Koga et al.[7].1,
L
-tryptophan aminotransferase; 2, indolelactate dehydrogenase; 3, indolepyruvate
decarboxylase; 4, indoleacetaldehyde oxidase.
Ó FEBS 2003 Structure–function studies of E. cloacae IDPC (Eur. J. Biochem. 270) 2323
10 m
M
magnesium sulphate, 1 m
M
EDTA, 5 m
M
dithio-
threitol, 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

Mes/NaOH pH 6.5, 1 m
M
dithio-
threitol, 0.25
M
ammonium sulphate. The fractions with the
highest catalytic activity and homogeneity were pooled,
quickly frozen in liquid nitrogen after addition of 0.2
M
ammonium sulphate, and stored at )80 °C.
SDS/PAGE
SDS/PAGE was carried out according to the method of
Laemmli [11]. Gels (10% (w/v) acrylamide) were stained
with Coomassie brillant blue G250.
Determination of enzyme concentration
The concentration of EcIPDC was determined spectro-
photometrically at 280 nm using a calculated molecular
absorption coefficient of
2
259 520
M
)1
Æcm
)1
[12]. ThDP-
containing samples were analysed using the method of
Bradford [13].
Syntheses of 4-substituted benzoylformates
Syntheses were performed according to Hallmann and
Ha

followed at 340 nm. Indolepyruvate was preincubated in
10 m
M
Mes pH 6.5 at 25 °C for 45 min to ensure the
generation of the ketone.
The ability of ScPDC and ZmPDC to decarboxylate
indolepyruvate was examined under the same conditions. In
the case of ZmPDC maximum enzyme concentration was
2.3 mgÆmL
)1
. Measurements with ScPDC were performed
at an enzyme concentration of 90 lgÆmL
)1
.
The plots of the reaction rate vs. substrate concentration
were fitted using the Michaelis–Menten equation in the case
of EcIPDC, or according to a substrate activation mech-
anism in the case of ScPDC [18]. For the substrate 4-NO
2
-
benzoylformate the kinetic constants were estimated from
the progress curves using the integrated Michaelis–Menten
equation.
Stopped-flow experiments were performed in 10 m
M
Mes
pH 6.5, 0.55 m
M
NADH, 450 UÆmL
)1

Mes pH 6.5, 50 m
M
Mg
2+
,0.35m
M
NADH, 1 UÆmL
)1
horse liver alcohol dehydrogenase, and 25 m
M
benzoyl-
formate as substrate at 366 nm. To obtain the K
d
of the
primary binding of ThDP the measurements were started
with the apoenzyme–magnesium complex (10.7 lgÆmL
)1
)at
20 °C. The progress curves were fitted according to Wang
et al. [19] with an equation containing an exponential and
a linear term.
One unit of catalytic activity is defined as the amount of
enzyme converting 1 lmol substrateÆmin
)1
.
1
H NMR experiments on indolepyruvate
To study the keto-enol tautomerism of indolepyruvate,
1
H NMR spectra of a solution of 1 m

¨
tz et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Boehringer Mannheim GmbH) and ZmPDC (244 kDa)
were used as molecular mass standards.
Small angle X-ray solution scattering with synchrotron
radiation. Data were collected on the X33 camera of the
European Molecular Biology Laboratory outstation at
Hasylab at the storage ring DORIS of the Deutsches
Elektronen Synchrotron (DESY) in Hamburg [20–23].
Measurements were performed at a camera length of
1.9 m using multiwire proportional chambers with delay
line readout [22] at a temperature of 12 °CandEcIPDC
concentrations of about 5 mgÆmL
)1
in 60 m
M
buffer at
different pH values (citrate pH 5.6, Mes pH 6.1, BisTris
pH 6.4, Pipes pH 6.8, Mops pH 7.2, Hepes pH 7.5,
Tricine pH 8.1, Bicine pH 8.3, borate pH 9.2, Ches
pH 9.5, and Caps pH 10.2), 62.5 m
M
ammonium sul-
phate, 3 m
M
dithiothreitol in the presence or absence of
10 m
M
ThDP/Mg
2+

D. I. Svergun, unpublished data). All protein concentra-
tions and pH values of the samples used for parameter
calculation were determined after the measurements.
Results
Purification of EcIPDC
The procedure, yielding the homogenous ThDP-free
enzyme, comprises four steps: streptomycin sulphate treat-
ment; ammonium sulphate precipitation; size exclusion
chromatography; and anion exchange chromatography.
After reconstitution of the holoenzyme the maximum
specific activity was % 1UÆmg
)1
using indolepyruvate as
substrate. EcIPDC is quite stable at 40 °C without any
further additions. A first-order rate constant of inactivation
of 10
)5
Æs
)1
was obtained in the elution buffer of the anion
exchange chromatography. Ammonium sulphate (0.2
M
)
stabilized the enzyme 14-fold. Further stabilization was
achieved by addition of ThDP/Mg
2+
. Addition of 10%
(v/v) glycerol had no effect. A molecular mass of 60 kDa per
subunit was determined by SDS/PAGE, corresponding to
the value calculated from the nucleotide sequence of the

demonstrated a pH-dependent equili-
brium between tetramers and dimers at lower pH and
dimers and monomers at higher pH. The presence of
cofactors strongly suppressed significant accumulation of
dimers (Fig. 2).
1
H NMR experiments on indolepyruvate
EcIPDC is unable to decarboxylate freshly prepared
solutions of indolepyruvate. Therefore, the chemical pro-
perties and purity of indolepyruvate were characterized by
1
H NMR spectroscopy. The
1
H NMR spectrum of freshly
dissolved indolepyruvate consists of the typical signals and
spin systems of the indole moiety (triplets of 5-H and 6-H at
7.12 and 7.18 p.p.m., doublets of 4-H and 7-H at 7.43 and
7.75 p.p.m and the singlet of 2-H at 7.81 p.p.m. with
identical integrals of all signals). The additional singlet of
the pyruvyl moiety at 6.65 p.p.m. with a relative integral of
1 with respect to the indole protons is consistent with the
occurrence of the enol form of indolepyruvate (Fig. 1). In
the course of the establishment of the equilibrium % 85% of
the enol form is converted into the ketone (half-time
% 8 min at 20 °C) as deduced from the appearance of
additional proton signals due to the indole part of
Fig. 2. pH dependence of the oligomeric state of EcIPDC. Volume
fractions were calculated from the scattering patterns with the program
OLIGOMER
in the absence of cofactors (A) and in the presence of 10 m

/K
m
value (330 s
)1
Æm
M
)1
) for the substrate
indole-3-acetaldehyde (data not shown) allowed application
of this assay. Under all conditions used, the reaction rate is
directly proportional to the EcIPDC concentration and
independent of the concentration of the auxiliary enzyme,
confirming that the coupled assay monitors the true rate of
EcIPDC catalysis. Figs 3 and 4 and Table 1 illustrate the
results of the steady-state kinetics for indolepyruvate,
pyruvate, benzoylformate, and 4-substituted benzoylfor-
mates (NO
2
-, Br-, Cl-, F-, C
2
H
5
-, CH
3
-, and CH
3
O-) as
substrates of EcIPDC. The enzyme has the highest catalytic
efficiency to the native substrate indolepyruvate, to 4-Cl-
benzoylformate and to 4-Br-benzoylformate (k

cat
(0.4 ± 0.01 s
)1
). Pyruvate has
Fig. 3. Dependence of the catalytic activity of EcIPDC on the concen-
tration of substituted benzoylformates (Bf) measured in 10 m
M
Mes
pH 6.5 at 30 °C. The lines represent the fits to hyperbolic kinetics.
Fig. 4. Dependence of the catalytic activity of EcIPDC on the substrate concentration measured in 10 m
M
Mes pH 6.5 at 30 °C. The lines represent
the fits to hyperbolic kinetics. Insets, corresponding stopped-flow progress curves. Straight lines are linear fits. Measurements were monitored at
340 nm for pyruvate and at 366 nm for the other substrates with a coupled optical test. Ipyr, indolepyruvate; Bf, benzoylformate; Pyr, pyruvate.
2326 A. Schu
¨
tz et al. (Eur. J. Biochem. 270) Ó FEBS 2003
the lowest affinity of all substrates investigated (K
m
3.38 m
M
).
The straight lines in the plots according to Hanes [26]
(data not shown) demonstrate that there is no indication
for any substrate activation processes in EcIPDC catalysis.
The absence of lag phases in the progress curves obtained
from stopped-flow experiments using indolepyruvate,
pyruvate, and benzoylformate as substrates for EcIPDC
at 30 °C (Fig. 4 insets) and 10 °C (data not shown)
confirm these results. However, a weak substrate excess

curves are presented in Fig. 6. The pseudo first-order rate
constants of reconstitution calculated from these time
courses show a hyperbolic dependence on the ThDP
concentration (at saturating Mg
2+
concentration), pointing
to a two-step mechanism of cofactor binding (Fig. 6 inset)
[27]. The calculated maximum rate constant of reconstitu-
tion is % 0.03 s
)1
and thus in the range of values determined
for other PDCs ([28]; J. Scha
¨
ffner
5,6
, unpublished data;
U. Mu
¨
cke
5,6
, unpublished data). A K
d
of 32.6 ± 4.6 l
M
determined for the binding of ThDP to EcIPDC is signifi-
cantly lower than that of other PDCs except ZmPDC [29].
Discussion
The purification procedure results in a homogenous ThDP-
free enzyme that is stabilized by the addition of 0.2
M

m
(l
M
)
K
m
(relative) k
cat
(s
)1
) k
cat (relative)
k
cat
/K
m
(s
)1
Æm
M
)1
)
Ipyr 20 ± 1.3 1.0 3.9 ± 0.07 1.0 199
Pyr 3381 ± 179 169.1 3.5 ± 0.08 0.9 1
Bf 1646 ± 32 82.3 46.4 ± 1.23 11.9 28
4-NO
2
-Bf 5 ± 0.5 0.25 0.4 ± 0.01 0.1 80
4-Cl-Bf 48 ± 2.0 2.4 5.3 ± 0.05 1.4 110
4-Br-Bf 19 ± 1.0 0.95 3.2 ± 0.03 0.8 168

and 25 °C within 20 min. Hydroxyphenylpyruvate and
phenylpyruvate behave in a similar manner [32]. Schwarz
and Bitancourt [33] demonstrated the tautomerism of
indolepyruvate by TLC. Our time-dependent
1
HNMR
measurements of aqueous solutions of indolepyruvate
confirm these results. After 20 min incubation at 20 °C,
85% of the substrate is present as ketone. The remaining
15% is probably responsible for the formation of highly
conjugated aromatic structures causing the well-known
reddish discoloration of aqueous solutions of the substance.
No EcIPDC activity is detectable with freshly prepared
solutions of indolepyruvate as substrate, but maximum
catalytic activity is obtained after incubation for about
45 min. Thus it can be concluded that only the ketone of
indolepyruvate is the substrate for the enzyme.
Application of a continuous optical assay for the steady-
state measurements modified according to Weiss et al.[17]
allowed detailed kinetic analysis of substrate specificity and
cofactor binding of EcIPDC. The enzymatic conversion of
all substrates studied in this work (pyruvate, the native
substrate of PDC, benzoylformate, the native substrate of
benzoylformate decarboxylase together with the 4-substi-
tuted derivatives, indolepyruvate, the native substrate of
IPDC) results in hyperbolic plots of catalytic activity vs.
substrate concentration (Figs 3 and 4). Corresponding
straight lines in Hanes plots (data not shown) and the
absence of lag phases in the stopped-flow time courses
(Fig. 4 insets) clearly demonstrates that there is no indica-

are comparable to that of the native substrate indolepyru-
vate. Both halogenations seem to mimic the best substrate
surrogates of indolepyruvate. The highest K
M
value and
the lowest specificity are found for pyruvate and only this
substrate displays a weak substrate excess inhibition (K
i
164 m
M
). The K
m
values determined for indolepyruvate and
pyruvate correspond to those found by Koga et al.[7]using
a discontinuous quantitative HPLC assay (15 l
M
and
2.5 m
M
, respectively). Interestingly, the K
m
value of pyru-
vate in EcIPDC catalysis is similar to that found for all
other PDCs and the same holds true for the weak substrate
excess inhibition. However, the corresponding k
cat
value of
EcIPDC is only about 2% of that of other PDCs.
Hammett [38] developed a method to calculate the
electronic effect of a substituent from studies on the

[17]. However, in ScPDC [40,41] all benzoylformates with
electron-withdrawing substituents exhibit a higher reaction
rate and all benzoylformates with electron-donating sub-
stituents have a lower one. EcIPDC binds all 4-substituted
benzoylformates with a higher affinity than the unsubsti-
tuted benzoylformate as is the case in ScPDC. With the
exception of 4-methoxybenzoylformate the substituted
benzoylformates have a lower affinity for benzoylformate
decarboxylase than does benzoylformate itself.
The hyperbolic dependence of the rate constants of
reconstitution, calculated from the corresponding progress
curves, on the concentration of ThDP (Fig. 6 inset) is
indicative of a two-step mechanism of cofactor binding as
Fig. 6. Progress curves of the reconstitution of EcIPDC with ThDP
measured by restoration of the catalytic activity of the formed holo-
enzyme for the substrate benzoylformate (25 m
M
)in10 m
M
Mes pH 6.5,
50 m
M
Mg
2+
,0.35m
M
NADH, and 1 UÆmL
-1
horse liver alcohol
dehydrogenase at 20 °C. The reaction was started with EcIPDC

resulting K
d
value of % 33 l
M
for the primary binding of
ThDP to the enzyme saturated with magnesium ions
illustrates a significantly higher affinity of the cofactor
ThDPtoEcIPDCthantoScPDCandPDCfromPisum
sativum (150–300 l
M
). Even a higher affinity was found for
ZmPDC [29].
A molecular mass corresponding to a tetramer of
EcIPDC at pH 6.0 was determined by two independent
methods, size exclusion chromatography and small angle
X-ray solution scattering. These results suggest that the
tetramer is stable in aqueous solution even without cofac-
tors and that this oligomeric state is catalytically active in
the presence of cofactors. Evaluation of the scattering
experiments with ThDP-free EcIPDC demonstrates a pH-
dependent equilibrium between tetramers, dimers and even
monomers. A similar behaviour (without occurrence of a
monomer fraction) was described for PDCs from various
organisms, but not for ZmPDC, where the tetramer is stable
from pH 5 to pH 9 [43]. The cofactors ThDP and Mg
2+
stabilize the tetrameric state of EcIPDC up to pH 7.5
(Fig. 2). A similar stabilization up to pH 8.5 was found for
ScPDC [44]. The quality of the scattering patterns allowed
the calculation of volume fractions of different oligomeric

M
) for indolepyruvate
and follows Michaelis–Menten kinetics, whereas ScPDC
exhibits sigmoid kinetics with a considerably lower affinity
for the substrate (S
0.5
¼ 0.7 m
M
) (Fig. 5). The S
0.5
values
for indolepyruvate and pyruvate (1.1 m
M
at pH 6.0;
J. Ermer
9
, unpublished data) are in the same range for
ScPDC.
As in ZmPDC and plant PDCs prominent amino acid
residues that restrict the size of the active site are conserved,
such as Trp392 and Trp551 (ZmPDC numbering), one can
assume that plant PDCs are also unable to accept indole-
pyruvate as substrate. Consequently, other pathways for the
biosynthesis of the phytohormone indoleacetic acid must
exist, not excluding the existence of a specific plant IPDC.
Yeast PDCs which do not possess such conserved space
filling amino acid residues, have a more open topology of
the substrate binding cavity and should thus presumably be
capable of using indolepyruvate as substrate, although with
a lower specificity than EcIPDC.

the 4-substituted benzoylformates vs. the substituent constants according
to Hansch et a l.[39].The k
cat
value corresponds to unmodified ben-
zoylformate.
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Ó FEBS 2003 Structure–function studies of E. cloacae IDPC (Eur. J. Biochem. 270) 2331