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

The yeast Kluyveromyces lactis formerly termed Saccharomyces lactis is able to assimilate lactose and Keywords allosteric enzyme activation; conformation equilibrium; disordered loop reg

Kluyveromyces lactis

Implications for the substrate activation mechanism of this enzyme Steffen Kutter1, Georg Wille1,*, Sandy Relle1, Manfred S Weiss2, Gerhard Hu¨bner1 and

Stephan Ko¨nig1

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 Mg2+ 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: koenig@biochemtech.uni-halle.de

*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 subsub-strate 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 lactispyruvate 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.

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 Mg2+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 substrsepar-ate 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 D2 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.

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 D2

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 Mg2+, 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

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)

Rr.i.m.(%) 8.0 (24.7)

Rp.i.m.(%) 3.5 (11.8) Overall B-factor from Wilson plot (A˚2) 28.3 Optical resolution (A ˚ ) 1.70

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)

Rfree(%) 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

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 subsub-unit B in Fig 1.

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.

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 Mg2+ 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 Mg2+ (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

Mg2+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.

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 Mg2+(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 Mg2+is presented as a green

sphere.

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 (Ka 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  5 A˚ [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

sediment was resuspended in a minimal volume of 0.1 m

Mes with 2 mm dithiothreitol, pH 6.0, loaded on a

Super-dexTM 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%

method of La¨mmli [29]) were combined, saturated with

the cofactors, 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 HitrapTM (GE Healthcare, Munich,

Germany) Sephadex columns (5· 5 mL) at 3 mLÆmin)1

The protein solution was loaded on a Poros20QE

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

(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,

sodium citrate, pH 6.45, 1 mm dithiothreitol, 5 mm ThDP,

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

8C 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

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 Rr.i.m. as well as the precision-indicating merging R-factor Rp.i.m.[31] were calculated using the pro-gram rmerge (available from http://www.embl-hamburg.de/

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

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 (http://www.biochem.ucl.ac.uk/bsm/PP/server)

Acknowledgements

The authors thank the EMBL outstation for access to beamline X11 at the DORIS storage ring, DESY, Hamburg

References

1 Schellenberger A, Hu¨bner G & Neef H (1997) Cofactor designing in functional analysis of thiamin diphosphate enzymes Methods Enzymol 279, 131–146

2 Hohmann S & Cederberg H (1990) Autoregulation may

control the expression of yeast pyruvate decarboxylase

structural genes PDC1 and PDC5 Eur J Biochem 188,

615–621

3 Davies DD (1967) Glyoxylate as a substrate for pyruvic

decarboxylase Proc Biochem Soc 104, 50P

4 Hu¨bner G, Weidhase R & Schellenberger A (1978)

The mechanism of substrate activation of pyruvate

decarboxylase: a first approach Eur J Biochem 92,

175–181

5 Hu¨bner G & Schellenberger A (1986) Pyruvate

decar-boxylase) potentially inactive in the absence of the

substrate Biochem Int 13, 767–772

6 Bringer-Meyer S, Schimz KL & Sahm H (1986)

Pyru-vate decarboxylase from Zymomonas mobilis Isolation

and partial characterization Arch Microbiol 146, 105–

110

7 Lu G, Dobritzsch D, Ko¨nig S & Schneider G (1997)

Novel tetramer assembly of pyruvate decarboxylase

from brewer’s yeast observed in a new crystal form

FEBS Lett 403, 249–253

8 Lu G, Dobritzsch D, Baumann S, Schneider G & Ko¨nig

S (2000) The structural basis of substrate activation in

yeast pyruvate decarboxylase) a crystallographic and

kinetic study Eur J Biochem 267, 861–868

9 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 thiamin

diphosphate-dependent enzyme pyruvate decarboxylase from the

yeast Saccharomyces cerevisiae at 2.3 A˚ resolution

J Mol Biol 256, 590–600

10 Baburina I, Gao Y, Hu Z, Jordan F, Hohmann S &

Furey W (1994) Substrate activation of brewer’s yeast

pyruvate decarboxylase is abolished by mutation of

cys-teine 221 to serine Biochemistry 33, 5630–5635

11 Baburina I, Dikdan G, Guo F, Tous GI, Root B &

Jor-dan F (1998) Reactivity at the substrate activation site

of yeast pyruvate decarboxylase Inhibition by distortion

of domain interactions Biochemistry 37, 1245–1255

12 Baburina I, Li H, Bennion B, Furey W & Jordan F

(1998) Interdomain information transfer during

sub-strate activation of yeast pyruvate decarboxylase The

interaction between cysteine 221 and histidine 92

Biochemistry 37, 1235–1244

13 Li H & Jordan F (1999) Effects of substitution of

tryp-tophan 412 in the substrate activation pathway of yeast

pyruvate decarboxylase Biochemistry 38, 10004–10012

14 Li H, Furey W & Jordan F (1999) Role of glutamate 91

in information transfer during substrate activation of

yeast pyruvate decarboxylase Biochemistry 38, 9992–

10003

15 Bianchi MM, Tizzani L, Destruelle M, Frontali L &

Wesolowski-Louvel M (1996) The ‘petite-negative’ yeast

Kluyveromyces lactishas a single gene expressing

pyru-vate decarboxylase activity Mol Microbiol 19, 27–36

16 Muller YA, Lindqvist Y, Furey W, Schulz GE, Jordan

F & Schneider G (1993) A thiamin diphosphate binding fold revealed by comparison of the crystal structures of transketolase, pyruvate oxidase and pyruvate decarboxy-lase Structure 1, 95–103

17 Pletcher J, Sax M, Turano A & Chang CH (1982) Effects of structural variations in thiamin, its derivatives and analogues Ann NY Acad Sci 378, 454–458

18 Dyda F, Furey W, Swaminathan S, Sax M, Farrenkopf

B & Jordan F (1993) Catalytic centers in the thiamin diphosphate dependent enzyme pyruvate decarboxylase

at 2.4 A˚ resolution Biochemistry 32, 6165–6170

19 Schenk G, Layfield R, Candy JM, Duggleby RG & Nixon PF (1997) Molecular evolutionary analysis of the thiamine-diphosphate-dependent enzyme, transketolase

J Mol Evol 44, 552–572

20 Dobritzsch D, Ko¨nig S, Schneider G & Lu G (1998) High resolution crystal structure of pyruvate decarboxy-lase from Zymomonas mobilis Implications for substrate activation in pyruvate decarboxylases J Biol Chem 273, 20196–20204

21 Jordan F, Nemeria N, Guo FS, Baburina I, Gao YH, Kahyaoglu A, Li HJ, Wang J, Yi JZ, Guest JR et al (1998) Regulation of thiamin diphosphate-dependent 2-oxo acid decarboxylases by substrate and thiamin di-phosphate.Mg(II)) evidence for tertiary and quaternary interactions Biochim Biophys Acta 1385, 287–306

22 Tittmann K, Golbik R, Uhlemann K, Khailova L, Schneider G, Patel M, Jordan F, Chipman DM, Dugg-leby RG & Hu¨bner G (2003) NMR analysis of covalent intermediates in thiamin diphosphate enzymes Biochem-istry 42, 7885–7891

23 Lie MA, Celik L, Jorgensen KA & Schiøtt B (2005) Cofactor activation and substrate binding in pyruvate decarboxylase Insights into the reaction mechanism from molecular dynamics simulations Biochemistry 44, 14792–14806

24 Liu M, Sergienko EA, Guo FS, Wang J, Tittmann K, Hu¨bner G, Furey W & Jordan F (2001) Catalytic acid– base groups in yeast pyruvate decarboxylase 1 Site-directed mutagenesis and steady-state kinetic studies on the enzyme with the D28A, H114F, H115F, and E477Q substitutions Biochemistry 40, 7355–7368

25 Krieger F, Spinka M, Golbik R, Hu¨bner G & Ko¨nig S (2002) Pyruvate decarboxylase from Kluyveromyces lac-tis) an enzyme with an extraordinary substrate activa-tion behaviour Eur J Biochem 269, 3256–3263

26 Svergun DI, Petoukhov MV, Koch MHJ & Ko¨nig S (2000) Crystal versus solution structures of thiamine diphosphate-dependent enzymes J Biol Chem 275, 297– 302

27 Konarev PV, Petoukhov MV & Svergun DI (2001) MASSHA) a graphics system for rigid-body modelling

of macromolecular complexes against solution scattering data J Appl Crystallogr 34, 527–532