Báo cáo khoa học: The crystal structure of pyruvate decarboxylase from Kluyveromyces lactis Implications for the substrate activation mechanism of this enzyme ppt

The crystal structure of pyruvate decarboxylase from
Kluyveromyces lactis
Implications for the substrate activation mechanism of this enzyme
Steffen Kutter
1
, Georg Wille
1,
*, Sandy Relle
1
, Manfred S. Weiss
2
, Gerhard Hu
¨
bner
1
and
Stephan Ko
¨
nig
1
1 Institute for Biochemistry, Department of Biochemistry & Biotechnology, Martin-Luther-University Halle-Wittenberg, Halle (Saale),
Germany
2 European Molecular Biology Laboratory Outstation, Hamburg, Germany
Pyruvate decarboxylase (PDC; EC 4.1.1.1) is a key
enzyme of carbon metabolism at the branching point
between aerobic respiration and anaerobic alcoholic
fermentation. It catalyzes the decarboxylation of pyru-
vate in plants, yeasts and some bacteria by using thi-
amine diphosphate (ThDP) and Mg
2+
as cofactors.

The catalytic cycle of ThDP enzymes is well estab-
lished [1] (Scheme 1). At first, the a-carbonyl group of
the substrate is attacked by the deprotonated C2 atom
of the thiazolium ring of ThDP [the ylid (I)]. In the
case of pyruvate, the resulting lactyl-ThDP (II) is sub-
sequently decarboxylated to yield the central interme-
diate of ThDP catalysis, the a-carbanion ⁄ enamine
(III). Protonation of III yields hydroxyethyl-ThDP
(IV), and the release of the second product acetalde-
hyde completes the catalytic cycle of ThDP.
The yeast Kluyveromyces lactis (formerly termed
Saccharomyces lactis) is able to assimilate lactose and
Keywords
allosteric enzyme activation; conformation
equilibrium; disordered loop regions;
thiamine diphosphate
Correspondence
S. Ko
¨
nig, Institute for Biochemistry,
Department of Biochemistry &
Biotechnology, Martin-Luther-University
Halle-Wittenberg, Kurt-Mothes-Str. 3,
06120 Halle (Saale), Germany
Fax: +49 345 5527014
Tel: +49 345 5524829
E-mail:
*Present address
Institute for Biophysics, Department of
Physics, Johann-Wolfgang-Goethe-University

Frankfurt ⁄ Main, Max-von-Laue-Str. 1,
60438 Frankfurt ⁄ Main, Germany
(Received 19 June 2006, accepted 13 July
2006)
doi:10.1111/j.1742-4658.2006.05415.x
The crystal structure of pyruvate decarboxylase from Kluyveromyces lactis
has been determined to 2.26 A
˚
resolution. Like other yeast enzymes,
Kluyveromyces lactis pyruvate decarboxylase is subject to allosteric sub-
strate activation. Binding of substrate at a regulatory site induces catalytic
activity. This process is accompanied by conformational changes and
subunit rearrangements. In the nonactivated form of the corresponding
enzyme from Saccharomyces cerevisiae, all active sites are solvent accessible
due to the high flexibility of loop regions 106–113 and 292–301. The bind-
ing of the activator pyruvamide arrests these loops. Consequently, two of
four active sites become closed. In Kluyveromyces lactis pyruvate decarb-
oxylase, this half-side closed tetramer is present even without any activator.
However, one of the loops (residues 105–113), which are flexible in nonacti-
vated Saccharomyces cerevisiae pyruvate decarboxylase, remains flexible.
Even though the tetramer assemblies of both enzyme species are different
in the absence of activating agents, their substrate activation kinetics are
similar. This implies an equilibrium between the open and the half-side
closed state of yeast pyruvate decarboxylase tetramers. The completely
open enzyme state is favoured for Saccharomyces cerevisiae pyruvate de-
carboxylase, whereas the half-side closed form is predominant for Kluyve-
romyces lactis pyruvate decarboxylase. Consequently, the structuring of the
flexible loop region 105–113 seems to be the crucial step during the sub-
strate activation process of Kluyveromyces lactis pyruvate decarboxylase.
Abbreviations

KlPDC, pyruvate decarboxylase from Kluyveromyces lactis; PDC, pyruvate decarboxylase; ScPDC, pyruvate decarboxylase from
Saccharomyces cerevisiae; ThDP, thiamine diphosphate.
FEBS Journal 273 (2006) 4199–4209 ª 2006 The Authors Journal compilation ª 2006 FEBS 4199
convert it to lactic acid. It is commercially utilized for
the production of recombinant chymosin, a proteolytic
enzyme used to coagulate milk in cheese manufac-
turing.
In contrast to S. cerevisiae, only one gene codes for
PDC in Kluyveromyces lactis. The protein (SwissProt
entry Q12629) has 86.3% identical residues and 96.4%
similar residues compared to SwissProt entry P06169,
the dominant PDC in S. cerevisiae [2]. It is known
from small-angle X-ray solution scattering experiments
(unpublished results) that the catalytically active form
of K. lactis PDC (KlPDC) is a homotetramer at micro-
molar protein concentrations (563 amino acid residues
per subunit, total molecular mass 240 kDa). The cofac-
tors ThDP and Mg
2+
are bound tightly, but not cova-
lently, at the interface of two monomers (Fig. 1). At
pH values > 8, the cofactors dissociate from the pro-
tein, resulting in complete loss of catalytic activity.
Lowering the pH to 5.7–6.3, which is also the opti-
mum for KlPDC catalysis, can restore this activity
almost completely.
In 1967, Davies [3] was the first to describe a sigmoi-
dal deviation of the plot of reaction rate vs. substrate
concentration for PDC from wheat germ. Hu
¨

bner
et al. [4] established a first model for this substrate
activation phenomenon. Stopped-flow kinetic tech-
niques were used to analyze the substrate activation of
S. cerevisiae PDC (ScPDC). From studies with the
inhibitor glyoxylic acid and the inconvertible activator
pyruvamide (2-oxopropane amide, the amide analog of
the substrate pyruvate), it was concluded that a separ-
ate binding site for the regulatory substrate molecule
must exist. Later, Hu
¨
bner and Schellenberger [5]
showed that the enzyme is potentially inactive in the
absence of substrate. With the single exception of the
bacterial enzyme from Zymomonas mobilis [6], all
PDCs studied so far are subject to substrate activa-
tion.
Lu et al. [7,8] described the structural consequences
of substrate activation on the basis of the crystal struc-
ture of pyruvamide-activated ScPDC compared to that
of ScPDC crystallized in the absence of any effectors
[9], which is assumed to be the nonactivated state of
the enzyme. Activation involves a rearrangement of
the two dimers within the tetramer: the D
2
symmetry
of the nonactivated ScPDC is broken, and an open
and a closed side of the tetrameric molecule is formed.
Two different binding sites of the activator were
located: one at the interface between the two domains

within one subunit, and one directly at the active site.
In the presence of pyruvamide, the loop regions 106–
113 and 292–301 undergo a disorder–order transition
and close over the active sites, thus possibly stabilizing
the binding of substrate.
An alternative pathway for substrate activation is
favored by Baburina et al. [10–12] and Li et al. [13,14],
who suggest that an activator molecule, bound to resi-
due Cys221, is the starting point for the activation
transition. However, no electron density for a bound
activator molecule could be detected directly at this
amino acid residue in pyruvamide-activated ScPDC.
Instead, pyruvamide was found to bind 10 A
˚
away
from Cys221, in a pocket formed by two of three
domains of the subunit [8].
Scheme 1. Catalytic cycle of pyruvate
decarboxylase. A prerequisite for substrate
binding at the cofactor thiamine diphosphate
(ThDP) is the deprotonation of the C2 atom
of the thiazolium ring (marked by an
asterisk). The resulting ylid of ThDP (I) can
attack the carbon atom of the carbonyl
group of the substrate pyruvate, generating
lactyl ThDP (II), the first tetrahedral
intermediate of the cycle. The subsequent
decarboxylation of II results in the central
reaction intermediate, the a-carbanion-
enamine of ThDP (III). Protonation of III

yields the second tetrahedral intermediate,
the hydroxyl ethyl ThDP (IV). Release of the
second product, acetaldehyde, completes
the cycle.
Crystal structure of pyruvate decarboxylase S. Kutter et al.
4200 FEBS Journal 273 (2006) 4199–4209 ª 2006 The Authors Journal compilation ª 2006 FEBS
Here, we describe the crystal structure of PDC from
the yeast K. lactis and the structural consequences of
the substrate activation of this PDC species. Our
model constitutes an extension to the activation model
previously proposed and established for ScPDC [8].
Results
Quality of the crystal structure model
The asymmetric unit contains a complete tetramer.
Hence, the final model consists of four polypeptide
chains arranged as a homotetramer of approximate D
2
symmetry. Each monomer was modeled using the
amino acid sequence deduced from KlPDC gene pdc1
[15], corresponding to SwissProt entry Q12629. The
refined model comprises residues 2–105, 114–289 and
303–562 of subunit A, residues 2–104 and 114–554 of
subunit B, residues 2–104 and 116–556 of subunit C,
residues 2–104 and 121–562 of subunit D, four mole-
cules of ThDP, four Mg
2+
, and 1649 water molecules.
The final R-factor is 0.158 (for complete data collec-
tion and processing statistics, see Table 1).
Fig. 1. Ca trace of the crystal structure

model of the Kluyveromyces lactis pyruvate
decarboxylase (KlPDC) tetramer. The four
subunits are colored individually (subunit A,
pink; subunit B, green; subunit C, blue;
subunit D, orange). The cofactors thiamine
diphosphate and Mg
2+
(presented in space-
filling mode, colored by their elements,
Mg
2+
in green) are located at the subunit
interface areas (A–B and C–D, respectively)
of both dimers. The open and the closed
side of the tetramer resulting from the spe-
cial dimer arrangement are indicated.
Table 1. Data collection and processing statistics. Values in paren-
theses correspond to the highest-resolution shell.
Number of crystals 1
Beamline X11
Detector MARCCD
Wavelength (A
˚
) 0.8125
Temperature (K) 100
Crystal–detector distance (mm) 180
Rotation range per image (°) 0.5
Total rotation range (°) 265.5
Space group P2
1

Unit cell parameters (A
˚
) a ¼ 78.72, b ¼ 203.09,
c ¼ 79.78, b ¼ 91.82°
Mosaicity (°) 0.40
Resolution limits (A
˚
) 99.0–2.26 (2.32–2.26)
Total number of reflections 549 432
Unique reflections 114 899
Redundancy 4.8
I ⁄ r (I) 20.2 (6.4)
Completeness (%) 98.5 (95.5)
R
merge
(%) 7.1 (21.5)
R
r.i.m.
(%) 8.0 (24.7)
R
p.i.m.
(%) 3.5 (11.8)
Overall B-factor from Wilson plot (A
˚
2
) 28.3
Optical resolution (A
˚
) 1.70
S. Kutter et al. Crystal structure of pyruvate decarboxylase

FEBS Journal 273 (2006) 4199–4209 ª 2006 The Authors Journal compilation ª 2006 FEBS 4201
Neither the terminal residues, nor residues 105–113
in all subunits and residues 290–302 in one subunit,
could be traced in the electron density map, prob-
ably because of too high flexibility of these regions.
Even in subunits B–D, in which the latter region
could be traced, the high flexibility of the loop is
evidenced by B-factors > 50 A
˚
2
, which are clearly
above the average of  22 A
˚
2
(Table 2). In the crys-
tal structure of nonactivated ScPDC, none of the
two loop regions are resolved [9]. However, they are
well defined in the structure of pyruvamide-activated
ScPDC [8]. Another flexible loop in KlPDC is the
one comprising amino acid residues 344–360. This
loop is located at the solvent-exposed surface of the
tetramer and it connects the middle and the C-ter-
minal domains (Fig. 2). In the crystal structure of
pyruvamide-activated ScPDC, the cleft between these
Table 2. Refinement statistics.
Resolution range (A
˚
) 23.58–2.26 (2.32–2.26)
Total number of atoms
(nonhydrogen)

18 466
Number of protein atoms 16 776
R
cryst
(%) 15.8 (16.3)
R
free
(%) 21.4 (27.0)
r.m.s.d. from ideality
Bonds (A
˚
) 0.015
Angles (°) 1.477
Ramachandran plot
% in most favored regions 92.5
Average B-factor (A
˚
2
)
Main chain 21.7
Side chain 22.9
Thiamine diphosphate 13.4
Mg
2+
15.8
Water molecules 29.7
Fig. 2. Ribbon representation of the Kluyveromyces lactis pyruvate decarboxylase (KlPDC) monomer. The domains are colored individually
(N-terminal PYR domain, red; middle R domain, green; C-terminal PP domain, blue; domain-connecting loops, yellow). The cofactors are
depicted in space-filling mode. The positions of the N-terminal and C-terminal amino acid residues of the model, the position of the flexible
loop region, which is omitted in the final model, and the position of the residues adjacent to the loop are labeled. The orientation of the sub-

unit is the same as that of subunit B in Fig. 1.
Crystal structure of pyruvate decarboxylase S. Kutter et al.
4202 FEBS Journal 273 (2006) 4199–4209 ª 2006 The Authors Journal compilation ª 2006 FEBS
domains contains the binding site for the activator
molecule.
Overall structure
The KlPDC tetramer consists of two asymmetrically
associated identical homodimers (r.m.s.d. < 0.41 A
˚
based on 7566 atoms). Although no activator is pre-
sent, the KlPDC tetramer contains an open and
a closed side and thus resembles more closely the
tetramer structure of pyruvamide-activated ScPDC
(Fig. 3) than that of the nonactivated ScPDC
(Fig. 4). In going from the nonactivated form of
ScPDC to the activated one, one dimer has to rotate
by about 30° relative to the other. For comparison,
the corresponding angle found for (nonactivated)
KlPDC is  36°. The main difference between KlPDC
and the activated form of ScPDC is the flexibility of
the loop regions 105–113 and 290–302. Whereas these
loops are completely ordered in pyruvamide-activated
ScPDC, residues 105–113 are completely disordered,
and 290–302 partially disordered, in KlPDC. As a
consequence, KlPDC resembles nonactivated ScPDC
more closely than activated ScPDC in terms of loop
flexibility.
Subunit structure
As in all other ThDP-dependent decarboxylases ana-
lyzed so far, the KlPDC subunit consists of three

domains (Fig. 2). According to Muller et al. [16], these
domains are termed the PYR domain (binding the am-
inopyrimidine ring of ThDP), the R domain (binding
regulatory effectors), and the PP domain (binding the
diphosphate residue of ThDP). All three domains exhi-
bit their typical a ⁄ b-topology. The central six-stranded
b-sheet of the PYR domain (residues 2–182) is sur-
rounded by seven a-helices. The R domain (residues
193–341) consists of five a-helices and a central six-
stranded b-sheet. A central six-stranded parallel
b-sheet and eight a-helices form the PP domain (resi-
dues 360–556). A superposition of ScPDC and KlPDC
monomers yields r.m.s.d. values < 0.85 A
˚
(based on
3650 aligned atoms). The largest displacements are
observed for the C-terminal helix (5.5 A
˚
) and for most
parts of the central R domain.
Fig. 3. Superposition of the main chain
atoms of tetramers of Kluyveromyces lactis
pyruvate decarboxylase (KlPDC) (pink) and
pyruvamide-activated Saccharomyces cere-
visiae PDC (ScPDC) (lime, PDB entry code
1QPB). The arrows indicate the loop regions
105–113 in each subunit, which are ordered
in pyruvamide-activated ScPDC and disor-
dered in KlPDC. The cofactors thiamine
diphosphate and Mg

2+
are shown in space-
filling mode. The closed and open sides of
the tetramers are indicated.
S. Kutter et al. Crystal structure of pyruvate decarboxylase
FEBS Journal 273 (2006) 4199–4209 ª 2006 The Authors Journal compilation ª 2006 FEBS 4203
Structure of the active site
The general architecture of the active site of KlPDC
corresponds to that of other ThDP-dependent
enzymes. Figures 1–3 illustrate the binding of the co-
factors ThDP and Mg
2+
at the interface between two
subunits. The aminopyrimidine ring of ThDP is bound
at the PYR domain of one subunit. The diphosphate
residue is bound to the PP domain of the other sub-
unit at the same dimer together with the octahedral
coordinated Mg
2+
(Fig. 5). The amino acid arrange-
ment at the active site enforces the so-called V-confor-
mation of ThDP [17]. This relative orientation of the
pyrimidine ring and the thiazolium ring is one of the
three conformations that occur in crystal structures of
isolated ThDP, but is the only one found in more than
60 crystal structures of ThDP-dependent enzymes ana-
lyzed so far. All residues at the active site in direct
vicinity to the cofactors are identical to those of
ScPDC. However, some side chain conformations
appear to be different. His114, which is thought to be

necessary for substrate and ⁄ or intermediate binding
[18–22], is adjacent to the disordered loop region 105–
113. Even the side chain of His114 exhibits rather poor
electron density. The c-carboxyl group of Asp28, a
residue important for reaction intermediate stabiliza-
tion [23], is shifted by about 2.5 A
˚
towards the C2
atom of the cofactor ThDP, when compared to pyruv-
amide-activated ScPDC. Some minor differences
(< 2 A
˚
) can be identified for residues Asn471, Thr475
and Glu477, which are involved in the binding of the
diphosphate group of ThDP, either directly or via
Mg
2+
coordination (Fig. 5).
Amino acid substitutions
Twenty of the 77 substitutions in KlPDC are noncon-
servative compared to ScPDC. Most of these residues
are located at the surface of the tetramer and are thus
probably not involved in the catalytic mechanism. No
Fig. 4. Comparison of the dimer arrangement within the tetramers of Kluyveromyces lactis pyruvate decarboxylase (KlPDC), Saccharomyces
cerevisiae PDC (ScPDC) and pyruvamide-activated ScPDC (PA-ScPDC). Tetramers (space-filling mode with individually colored subunits) are
represented in three different orientations; the modes of 90° rotation are indicated as well as the angles resulting from dimer rearrange-
ments in KlPDC and PA-ScPDC.
Crystal structure of pyruvate decarboxylase S. Kutter et al.
4204 FEBS Journal 273 (2006) 4199–4209 ª 2006 The Authors Journal compilation ª 2006 FEBS
exchanges occur at the active site or the putative regu-

latory site [8]. Three substitutions (Asn143-Ala,
Ala196-Ser, and Ser318-Asn, the first residue referring
to KlPDC and second residue to ScPDC) are located
directly at the dimer–dimer interface (Fig. 6). These
might affect the dimer–dimer interactions, but none of
them are located at the monomer–monomer interface
within the dimers. Two exchanges (Val104-Ile and
Ser106-Ala) can be found in the flexible loop region
105–113.
Discussion
The structural basis of the activation of PDC is the
rotation of one dimer relative to the other within the
tetramer. This rotation is accompanied by local
Fig. 5. Stereo view of the active site in Kluyveromyces lactis pyruvate decarboxylase (KlPDC). Residues in the vicinity (5 A
˚
cut-off) of the co-
factors thiamine diphosphate (ThDP) (presented in stick mode, colored by the elements) and Mg
2+
(green sphere) are shown. Amino acid
residues of the PYR domain of one subunit are shown and labeled in red, and those of the PP domain of the other subunit within the same
dimer in blue. Residues are presented in stick mode; those with different orientations in KlPDC and pyruvamide-activated Saccharomyces
cerevisiae pyruvate decarboxylase (ScPDC) are presented in ball-and-stick mode (with gray background of the labels). A green asterisk and
an arrow indicate the C2 atom of ThDP, the substrate-binding site.
Fig. 6. Location of amino acid residues
resulting from nonhomologous exchanges in
Kluyveromyces lactis pyruvate decarboxy-
lase (KlPDC) compared to Saccharomyces
cerevisiae PDC (ScPDC) at the dimer inter-
face of the tetramer. Subunits (Ca trace)
together with their highlighted residues and

labels are colored individually. Cofactors are
presented in stick mode, colored by the ele-
ments, and Mg
2+
is presented as a green
sphere.
S. Kutter et al. Crystal structure of pyruvate decarboxylase
FEBS Journal 273 (2006) 4199–4209 ª 2006 The Authors Journal compilation ª 2006 FEBS 4205
conformational changes within the subunits due to the
binding of pyruvamide between the R and the PP
domains. The dimer reorientation leads to the genera-
tion of a closed and an open side in the tetramer. Con-
sequently, new interaction areas are formed at the
closed side of the molecule. The most important one is
a disorder––order transition of two loop regions,
which are flexible in the nonactivated state (residues
106–113 and 292–301). These loops close over the act-
ive sites and shield the catalytic centers from the sol-
vent. In accordance with these results, Liu et al. [24]
have suggested the involvement of two histidine resi-
dues (His114 and His115) adjacent to loop 106–113 in
substrate and ⁄ or intermediate binding, based on kin-
etic studies of ScPDC variants. In contrast to the
situation observed for ScPDC, a half-side closed
quaternary structure of the tetramer of KlPDC exists
already in the nonactivated state. This observation,
based on the crystal structure, is corroborated by
small-angle X-ray scattering experiments that reveal a
more compact structure of nonactivated KlPDC
(radius of gyration 3.85 nm) compared to nonactivated

ScPDC (radius of gyration 3.95 nm) (unpublished
results). Furthermore, solution structure models calcu-
lated ab initio from small-angle X-ray scattering data
at low resolution (> 2 nm) illustrate a nonplanar
dimer arrangement in the KlPDC tetramer.
The observed differences in the three-dimensional
structure of both yeast PDCs manifest themselves in
the quaternary arrangement only. The monomers and
dimers can be superimposed with relatively low
r.m.s.d. values, which is to some extent expected
because of the high homology of their amino acid
sequences. However, it was shown previously that dif-
ferences exist between the two enzymes based on
detailed kinetic studies of KlPDC substrate activation
[25]. Analyses of the microscopic rate constants for
this process in various PDCs illustrated a particularly
low binding affinity for the substrate at the regulatory
site (K
a
value) in the case of KlPDC. The half-side
closed structure of the KlPDC tetramer may reflect
this special kinetic behavior. In the case of KlPDC,
binding of the regulatory substrate is not required for
the induction of a change in the dimer assembly as in
pyruvamide-activated ScPDC ) this conformation is
already preformed. From a structural point of view, it
is in fact possible that substrate activation of KlPDC
involves only a part of the processes in ScPDC,
namely, binding of the regulatory substrate(s) in the
cleft between the R and PP domains. The bound

activator may then enhance the rigidity of the
enzyme molecule and drive the disorder–order trans-
ition of the flexible loop region (residues 105–113).
This loop forms a lid over the active site, making it
inaccessible to solvent and thereby allowing the cata-
lytic reaction [8].
The substrate activation model for KlPDC has been
developed on the basis of crystal structure models
only. One can argue that solution structures may differ
from these models and that crystal contacts may influ-
ence interactions of neighboring molecules. However,
we believe that our interpretation is supported by the
similarity of the quaternary structures of KlPDC and
pyruvamide-activated ScPDC, although the first has
been crystallized in the absence of any allosteric effec-
tors and the latter in the presence of high concentra-
tions of the substrate surrogate pyruvamide.
Furthermore, we have previously shown that crystal
and solution structures of several ThDP-dependent
enzymes are essentially identical in the absence of
effectors [26]. Differences seem to be dependent on the
compactness of the enzyme molecules. The dimer
arrangement in the crystal structure of tetrameric non-
activated ScPDC is rather loose (dimer interface area
1640 A
˚
2
compared to 2700 A
˚
2

calculated for KlPDC,
and 3200 A
˚
2
for pyruvamide-activated ScPDC). A
nonactivated ScPDC model with an altered dimer
assembly within the tetramer resulted from rigid body
refinement [27] of crystallographic vs. solution scatter-
ing data. In this solution structure, the dimers of the
crystal structure are rotated  15° and their distance is
decreased by  5A
˚
[26]. Possibly, an equilibrium
between various quaternary PDC structures exists,
which is shifted more towards a planar dimer orienta-
tion in ScPDC and towards the half-side closed con-
formation in KlPDC, perhaps due to the amino acid
substitutions at the dimer interface mentioned above.
Binding of the regulatory substrate may then stabilize
the latter conformation in KlPDC and enable effective
catalysis.
Experimental procedures
Protein expression and purification
Protein expression and purification for both species was
carried out according to Sieber et al.(ScPDC [28]), and
Krieger et al.(KlPDC [25]), with some modifications.
ScPDC
The protamine sulfate treatment was omitted. Precipitation
ranges were changed: for acetone (55–70%, v ⁄ v) and for
ammonium sulfate (29.25–30.75 g per 100 mL). An addi-

tional ammonium sulfate precipitation of the protein guar-
anteed the removal of all traces of acetone. The resulting
Crystal structure of pyruvate decarboxylase S. Kutter et al.
4206 FEBS Journal 273 (2006) 4199–4209 ª 2006 The Authors Journal compilation ª 2006 FEBS
sediment was resuspended in a minimal volume of 0.1 m
Mes with 2 mm dithiothreitol, pH 6.0, loaded on a Super-
dex
TM
200 column (26 · 600 mm), and eluted with the
same buffer, but with 0.3 m ammonium sulfate at a flow
rate of 0.5 mLÆmin
)1
. Fractions with catalytic activities
above 35 UÆmg
)1
(1 U is defined as the consumption of
one lmol of substrate per min) and with more than 95%
homogeneity (judged by SDS ⁄ PAGE according to the
method of La
¨
mmli [29]) were combined, saturated with
the cofac tors, and precipitated with solid ammonium sulfate.
The pellets were stored at ) 20 °C after quick freezing.
KlPDC
Here, the range used for ammonium sulfate precipitation
was 28.5–34.75 g per 100 mL. Size exclusion chromatogra-
phy was performed as described above for ScPDC. Frac-
tions were combined, precipitated with ammonium sulfate,
resuspended in 20 mm Bistris, pH 6.8, with 2 mm dithiothre-
itol, and desalted on Hitrap

TM
(GE Healthcare, Munich,
Germany) Sephadex columns (5 · 5 mL) at 3 mLÆmin
)1
.
The protein solution was loaded on a Poros20QE (Persep-
tive Biosystems GmbH, Wiesbaden, Germany) anion
exchange column (4.6 · 100 mm) in the same buffer and
eluted with an ammonium sulfate gradient of 0–500 mm at
a flow rate of 2 mLÆmin
)1
. Fractions with catalytic activities
above 40 UÆmg
)1
and with more than 95% homogeneity
(according to SDS ⁄ PAGE) were combined, saturated with
the cofactors, and precipitated with solid ammonium sul-
fate. The pellets were stored at ) 20 °C after quick freezing.
Crystallization
KlPDC, stored as frozen ammonium sulfate precipitate,
was diluted in crystallization buffer (50 mm Mes, pH 6.45,
5mm ThDP, 1 mm dithiothreitol, 5 mm MgSO
4
or 35 mm
sodium citrate, pH 6.45, 1 mm dithiothreitol, 5 mm ThDP,
5mm MgSO
4
). Excess ammonium sulfate was removed,
and the protein was concentrated by the use of centrifugal
concentrators (0.5 mL, 30 kDa cut-off). KlPDC was crys-

tallized by hanging drop vapor diffusion in 24-well cell
culture plates. Four-microliter drops of protein solution (3–
15 mgÆmL
)1
) were mixed 1 : 1 with PEG 2000 ⁄ PEG 8000
(12–24%, w ⁄ v) in crystallization buffer. The best crystals
were obtained at 20% (w ⁄ v) PEG and 2 mg KlPDC ⁄ mL at
8 °C. Microcrystals in Mes buffer were obtained after
3 days. Larger single crystals (0.4 · 0.02 · 0.02 mm)
appeared after about 4 weeks. These crystals were stable
for several months. Microcrystals in citrate buffer could be
detected after 24 h. They grew to a final size of
0.6 · 0.02 · 0.02 mm over 10 days, with higher reproduci-
bility than those grown in Mes buffer. However, these crys-
tals were stable for 2 weeks only and disintegrated in
solutions with PEG or glycerol concentrations less than
12% (w ⁄ v).
Data collection
Data were collected under cryogenic conditions from a single
crystal of KlPDC, grown in Mes buffer. The crystal was
soaked with a cryoprotectant containing reservoir solution
with 15% (v ⁄ v) glycerol for 30 s, frozen in liquid nitrogen
and transferred into the cryogenic nitrogen stream at the
beamline. A native dataset was recorded on beamline X11
(EMBL, Hamburg, Germany) using an MARCCD detector.
The data were indexed and integrated using denzo and
scaled using scalepack [30]. The redundancy-independent
merging R-factor R
r.i.m.
as well as the precision-indicating

merging R-factor R
p.i.m.
[31] were calculated using the pro-
gram rmerge (available from />msweiss/projects/msw_qual.html or from MSW upon
request). Intensities were converted to structure factor ampli-
tudes using the program truncate [32,33]. Table 1 summar-
izes the data collection and processing statistics. The optical
resolution was calculated using the program sfcheck [34].
Structure solution and refinement
Initial phases were obtained from the model of the ScPDC
dimer (PDB entry code 1QPB) by molecular replacement
with the program molrep [32]. The search model for this
procedure was generated by automated modeling of the
KlPDC amino acid sequence using the swissmodel mode-
ling server [35]. Refinement (rigid body, TLS and
restrained) was carried out against this data set using the
program refmac5 [32]. Inspection of electron density,
model building and checking was done with the program
coot [36]. Several cycles of refinement and manual model
building were carried out until the free R-factor and the
crystallographic R-factor had converged (Table 2).
Figures were prepared with pymol (DeLano Scientific,
San Carlos, CA) and ds viewerpro (Accelrys Software
Inc., San Diego, CA). The coordinates and structure factors
have been deposited in the Protein Data Bank, http://
www.pdb.org (PDB ID code 2G1I).
Interface-accessible surface areas were calculated by using
the program provided by the protein–protein interaction
server ( />Acknowledgements
The authors thank the EMBL outstation for access to

beamline X11 at the DORIS storage ring, DESY,
Hamburg.
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S. Kutter et al. Crystal structure of pyruvate decarboxylase
FEBS Journal 273 (2006) 4199–4209 ª 2006 The Authors Journal compilation ª 2006 FEBS 4209