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: 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 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 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 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). 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The crystal structure of pyruvate decarboxylase from Kluyveromyces lactis Implications for the substrate activation mechanism of this enzyme Steffen Kutter 1 , Georg. 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. 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