From the Department of Medical Biochemistry and
Biophysics, Karolinska Institutet S-17177 Stockholm, Sweden and the
§ Institut für Biochemie, Fachbereich
Biochemie/Biotechnologie 1Martin-Luther-Universität
Halle-Wittenberg, D-06099 Halle/Saale, Germany
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ABSTRACT |
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The crystal structure of tetrameric pyruvate
decarboxylase from Zymomonas mobilis has been determined at
1.9 Å resolution and refined to a crystallographic
R-factor of 16.2% and Rfree of
19.7%. The subunit consists of three domains, all of the /
type.
Two of the subunits form a tight dimer with an extensive interface
area. The thiamin diphosphate binding site is located at the
subunit-subunit interface, and the cofactor, bound in the V
conformation, interacts with residues from the N-terminal domain of one
subunit and the C-terminal domain of the second subunit. The 2-fold
symmetry generates the second thiamin diphosphate binding site in the
dimer. Two of the dimers form a tightly packed tetramer with pseudo 222 symmetry. The interface area between the dimers is much larger in
pyruvate decarboxylase from Z. mobilis than in the yeast
enzyme, and structural differences in these parts result in a
completely different packing of the subunits in the two enzymes. In
contrast to other pyruvate decarboxylases, the enzyme from Z. mobilis is not subject to allosteric activation by the substrate.
The tight packing of the dimers in the tetramer prevents large
rearrangements in the quaternary structure as seen in the yeast enzyme
and locks the enzyme in an activated conformation. The architecture of
the cofactor binding site and the active site is similar in the two
enzymes. However, the x-ray analysis reveals subtle but significant
structural differences in the active site that might be responsible for
variations in the biochemical properties in these enzymes.
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INTRODUCTION |
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Pyruvate decarboxylase (PDC)1 is a key enzyme in alcohol fermentation and depends on thiamin diphosphate (ThDP) and Mg(II) ions for catalytic activity. The enzyme catalyzes the conversion of pyruvate to acetaldehyde and carbon dioxide but is also able to utilize other 2-oxo acids as substrates. The obligatory fermentative Gram-negative bacterium Zymomonas mobilis uses only hexoses as carbon sources for glycolysis and produces ethanol and carbon dioxide via the Entner-Doudoroff pathway (1). In this organism, PDC amounts to 4% of the total soluble protein and to 10% of the extractable protein after cell lysis (1). There appears to be only one gene coding for this enzyme in Z. mobilis (2).
In solution, native ZmPDC is a tetramer of four identical subunits, and each subunit consists of 568 amino acids with a molecular mass of about 60 kDa (1, 2). Every subunit binds a set of cofactors (ThDP and Mg(II) ions) very tightly but not covalently at pH 6.0, the optimum for catalytic activity. The mechanism of cofactor binding in ZmPDC is similar to that for the yeast enzyme (1, 3). The cofactors stabilize the quaternary structure in a wide range of pH from 4.6 to 8.5, but more alkaline conditions lead to complete loss of catalytic activity because of dissociation of the cofactors (4).
A common feature of pyruvate decarboxylases, with the exception of ZmPDC, is their allosteric regulation by the substrate or other activator molecules such as pyruvamide (5, 6). Crystallographic studies of yeast PDC revealed considerable differences in the tetramer assembly between enzyme species obtained in the absence (7, 8) or presence of the activator pyruvamide (9). Cross-linking and small angle x-ray solution scattering experiments also indicated significant tetramer reassembly and conformational changes during substrate activation in yeast PDC (10-12).
Here, we present the crystal structure of recombinant PDC from Z. mobilis at 1.9 Å resolution. The crystallographic study reveals a novel, as yet unobserved tetramer assembly in pyruvate decarboxylases. Comparison of the quaternary structures of PDC from Z. mobilis and yeast suggests that the structural differences in the interface regions might be related to the differences in their kinetic behavior. In addition, the crystallographic analysis provides further insights into the structural basis of catalysis and substrate specificity in pyruvate decarboxylases.
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MATERIALS AND METHODS |
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Expression and Purification--
The gene coding for ZmPDC was
expressed in the Escherichia coli SG13009 prep4 strain
containing the Z. mobilis gene ATCC 29191. This strain was
kindly provided by Dr. Martina Pohl (Institut für
Enzymtechnologie, Heinrich-Heine-Universität Düsseldorf). Expression of the PDC gene was induced by addition of 0.5 mM isopropyl -D-thiogalactosylpyranoside in
the late exponential growth phase. The cells were harvested by
centrifugation and disrupted in a French Press (SLM Instruments, Inc.).
Ammonium sulfate precipitation was carried out in two steps (30 and
42% w/v ammonium sulfate, respectively). The precipitate from the last
step, which contains crude pyruvate decarboxylase, was suspended in 10 mM Mes/NaOH, pH 6.5, 1 mM MgSO4,
0.2 mM ThDP, 1 mM dithioerythitol and dialyzed overnight against the same buffer. The enzyme solution was applied to
Fraktogel EMD TMAE (S) (Merck, column 2.6 × 10 cm) equilibrated with the same buffer without ThDP and MgSO4 (flow rate 1 ml/min). The protein was eluted with a linear ammonium sulfate gradient (0-40 mM in 100 ml) at 4 °C. The purest fractions
showed PDC activity of 100-120 units/mg and about 95% homogeneity in
SDS-polyacrylamide gel electrophoresis. These fractions were collected
and concentrated to 30-40 mg/ml by ultrafiltration. Simultaneously,
the buffer was changed to 10 mM sodium citrate pH 6.0. The
concentrated protein solution was stored at
20 °C without
significant loss of activity.
Crystallization-- ZmPDC was crystallized using the hanging drop vapor diffusion method. Droplets were set up for crystallization by mixing 4 µl of solution containing 13 mg/ml protein and 1 mM dithioerythitol with 4 µl of the reservoir solution containing 100 mM Mes/NaOH, pH 6.5, and 24% (w/v) PEG 1500. Tiny crystals were obtained within 10 days at 20 °C. Streak seeding was used to improve crystal size. The above protein solution containing 5 mM ThDP, 5 mM MgSO4, and 1 mM dithioerythitol was mixed with reservoir solution of 100 mM sodium citrate, pH 6.0, and 19-22% (w/v) PEG 1500 and pre-equilibrated for 2 days. After seeding, crystals appeared within a few hours and grew to a maximum size of 0.7 × 0.5 × 0.2 mm in 3 days.
Data Collection--
The crystals were soaked in a solution
containing 100 mM sodium citrate, pH 6.0, 22.5% (w/v) PEG
1500 and 17% (v/v) glycerol for 5 min and transferred into a cryogenic
nitrogen gas stream at 110 K. The x-ray diffraction data sets were
collected on a MAR research image plate mounted on a Rigaku rotating
anode, operating at 50 kV and 90 mA. Data processing was carried out by
the DENZO/SCALEPACK packages (13). The crystals belong to the triclinic
space group P1 with cell dimensions a = 69.9 Å,
b = 92.0 Å, and c = 98.0 Å, = 103.7 °,
= 94.5 °,
= 112.3 °. There are four ZmPDC
monomers in one asymmetric unit, resulting in a packing density of 2.7 Å3/Da. Details of the data collection are given in Table
I.
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Structure Solution by Molecular Replacement-- The structure of ZmPDC was determined by molecular replacement using a model of form B2 ScPDC refined at 2.4 Å (9),3 which shares only 28% sequence identity with ZmPDC. Orientation and positions of the molecules were determined using the AMORE program (14) with a yeast PDC dimer as a search model. Both self- and cross-rotation functions were calculated with x-ray data in the resolution interval 10-3 Å with an integration radius of 30 Å. Two orientation solutions were found with correlation coefficients 0.125 and 0.118, respectively (1.8 times the highest noise peak). With the position of one dimer fixed in the P1 space group, a cross-translation function using the data in the 10-3 Å resolution range determined the relative position of the other dimer with a correlation coefficient of 0.173 (1.3 times the highest noise peak) and an R-factor of 0.52.
Model Building and Crystallographic Refinement--
A test set
of 4% of the reflections was excluded before starting any
crystallographic refinement to monitor the Rfree
value. Rigid body refinement was carried out with XPLOR (15) using data
in the 15-2.4 Å resolution interval. The resulting model (Rfree = 0.50) was used for NCS averaging with
the Dm program (16) in the CCP4 package (17) and the Rave packages
(18). Based on the averaged electron density maps, the model was
rebuilt using the O program (19), and the ZmPDC sequence was fitted to
the electron density map. Atomic positions and B-factors of the model
were refined with noncrystallographic symmetry restraints using REFMAC
(20), and the model was rebuilt according to the resulting averaged
electron density map. Iterations of the procedure were performed with
the resolution gradually extended to 1.86 Å. When the
Rfree value had dropped to 27.5% and most of
protein atoms were defined, 2Fo Fc and Fo
Fc maps were used for further inspection of the
model. NCS restraints were released for several residues as indicated
by the electron density map. Most water molecules were added
automatically using the PEAKMAX program in CCP4 (17), the PEAKCHECK
program (written by J. Smith), and the WATNCS
program.4 NCS restraints were
introduced for most of the water molecules in the refinement.
Additional solvent molecules that did not follow NCS were added by
visual examination of the electron density map in the final stage.
Errors in the model were found automatically from
Fo
Fc maps by the
DIFLIST program5 and
corrected on the display using O. At this stage, it became obvious that
the bound cofactor had undergone chemical degradation in the ZmPDC
crystals, and a model of an ThDP analogue with an open thiazolium ring
was introduced. Citrate molecules were modelled as well as double
conformations for some of the amino acid residues. Statistics of the
refinement and the final protein model are given in Table
II.
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Structural Analysis-- The quality of the model was examined using PROCHECK (21). Structural comparisons of ZmPDC with form A ScPDC (PDB accession code 1PVD) (8), form B ScPDC (PDB accession code 1YPD) (9), and pyruvate oxidase (PDB accession code 1POX) (22) were carried out using the TOP program (23). The solvent-accessible surface was analyzed with the VOIDOO (24) and the AREAIMOL (17) programs.
Activity Measurements--
Enzymatic activity was assayed at
30 °C and 340 nm with a NADH/ADH coupled test in 100 mM
sodium citrate, pH 6.1, according to Holzer et al. (25). One
unit of activity is the quantity of enzyme that catalyzes the formation
of 1 µmol of product/min. The protein concentration was determined
spectrophotometrically at 280 nm ( = 275 320 M
1 cm
1).
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RESULTS |
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Structure Determination and Model Quality-- The crystal structure of ZmPDC was determined by molecular replacement. Electron density maps calculated from crude models at different stages of the refinement allowed tracing of the polypeptide chains, even for those parts where the ZmPDC chain is quite different in structure compared with ScPDC. The final model of ZmPDC in the asymmetric unit is a homotetramer of four monomers related by pseudo 222 symmetry. The model contains 4 × 565 amino acids, (comprising residues 2-566), four chemically modified ThDP molecules, four Mg(II) ions and four citrate molecules, and a total of 2569 water molecules. Most of these solvent molecules (2448) fully or partially follow the pseudo symmetry.
The structure of ZmPDC was refined to an R-factor of 16.2% and an Rfree value of 19.7% using all the data between 15-1.86 Å with good stereochemistry (for detailed statistics see Table II). The electron density map was of very good quality (Fig. 1) and allowed location of almost all protein atoms, except the N-terminal methionine and the two C-terminal residues. Electron densities were poor or weak for side chain atoms of several residues, including Glu227 and Asp530 in all subunits, Lys523 in three and Glu520 in one of the subunits. All of these residues are exposed to solvent on the protein surface. Double conformations were found for two residues in all the subunits, Lys553 located on the protein surface and Ile472 at the active site.
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Overall Structure--
ZmPDC is a homotetramer with an overall
size of approximately 85 × 98 × 118 Å. Each monomer can be
divided into three domains, denoted as PYR (residues 1-188), R
(residues 189-354), and PP (residues 355-568)
domains,6 each with an open
/
topology (Fig. 2). The
nomenclature of the secondary structural elements is shown in Figs. 2
and 4B. No significant structural differences were found
between the subunits, except for the side chains of about 20 residues,
which are involved in the crystal packing.
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ThDP Binding-- The four identical subunits, in combination with the pseudo 222 symmetry of the tetramer, generate four cofactor and substrate binding sites in the ZmPDC molecule. The ThDP and substrate binding sites are located in narrow clefts at the interfaces formed by the PYR domains from one subunit and the PP domains of another subunit. The ThDP binding site is deeply buried inside the molecule, about 15 Å away from the protein surface. The cofactor is bound in the V conformation as found in other ThDP-dependent enzymes such as ScPDC (7), transketolase (31), and POX (22). The pyrimidine ring of ThDP interacts with the PYR domain of one subunit, whereas the residual part interacts with the PP domain of another subunit. The Mg(II) ion anchors the diphosphate group of ThDP to the protein, and it forms an octahedral coordination sphere with two oxygen atoms of the diphosphate group of ThDP, the side chain oxygen atoms of Asp440 and Asn467, the main chain oxygen atom of Gly469, and a water molecule (Fig. 3).
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Citrate Binding Sites-- During the crystallographic refinement, strong electron density that did not represent any protein atoms was found at the dimer-dimer interface. The shape of the electron density and the high concentration of buffer ions lead us to interpret this residual electron density as citrate molecules. The four citrate ions might contribute to the tetramer assembly by electrostatic interactions and hydrogen bonds to protein side chains. The three carboxyl groups of each citrate molecule form salt bridges with five residues from two subunits, His150, Lys153, and Arg157 from one subunit and Arg310 and Arg318 from another subunit. In addition, several water molecules link citrate and protein atoms through hydrogen bonds, providing further stabilizing interactions.
Comparison with Yeast Pyruvate Decarboxylase--
The overall
topology of the subunit of ZmPDC is very similar to that of ScPDC.
However, the orientation between the three domains is slightly
different to the orientation observed in the two forms of ScPDC, and
these relative shifts correspond to rotations of approximately
6-8 ° (Table III). Furthermore,
considerable differences were found in the number of secondary
structural elements. When superposing the individual domains between
the two enzymes, it was found that 7 of the 24 -helices in ZmPDC
differ considerably with respect to length and orientation from their
counterparts in ScPDC (Fig. 4). In
general, structural differences are significantly higher for the R
domains than those found for the other two domains. The twist of the
-sheet in this domain is quite different in the two enzyme species,
probably because of large differences of amino acid compositions and
structures in the loop regions.
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DISCUSSION |
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Tetramer Assembly and Substrate Activation-- In contrast to other pyruvate decarboxylases, ZmPDC does not show allosteric activation by the substrate (1, 36). In ScPDC, substrate analogues such as pyruvamide (5) can act as substitutes in the activation process. Chemical modification (37) implicated cysteinyl side chains in the activation process. Crystallography (7, 8) and site-directed mutagenesis (38) subsequently suggested Cys221 of the R domain of ScPDC as the activator binding site, possibly through the formation of a covalent hemithioketal with the activator molecule. A signal transducing pathway from the R domain to the active site of the enzyme has been proposed through which the formation of this adduct could be translated into conformational changes at the active site (8). Indeed, crystallization of yeast PDC in the presence of the activator pyruvamide (9) and ketomalonate (39), respectively, revealed large conformational changes in the enzyme, involving both loop closure at the active site and tetramer reassembly. Surprisingly, in neither study was electron density for an activator molecule found close to Cys221. Despite ambiguities about which (if any) of the structures of the yeast enzyme represents the activated enzyme, it is nevertheless clear that ScPDC is able to readily undergo large conformational changes and that these changes are triggered by molecules acting as activators (7-11, 30, 39).
There is, however, a striking difference in the extent of the interface between the dimers in both forms of the yeast enzyme on the one hand and the dimers in ZmPDC on the other hand. The extensive interface region in ZmPDC, which also may account for the higher stability of ZmPDC compared with the yeast enzyme (4), makes large conformational changes during catalysis very unlikely. The only conformational changes required in ZmPDC during catalysis invoke the C-terminal helix, which has to swing out of the way to allow access of the substrate to the active site. After binding of the substrate, this helix might close the active site to create a hydrophobic environment, which would facilitate the enzymatic reaction. However, because this helix is at the surface of the enzyme, it does not need to involve other subunits. The activity of ZmPDC is higher than that of yeast PDCs by about 3-fold (1), and this difference in turnover could be related to the magnitude of the conformational changes occurring in the two enzymes during catalysis.Active Site and Catalysis-- Although the enzyme is observed in a closed form with restricted access to the active site, there is a sizeable cavity close to the thiazolium ring of the cofactor that is assumed to be the binding site for pyruvate. Model building of the central intermediates, 2-(2-hydroxypropionyl) ThDP and 2-(1-hydroxyethyl) ThDP, respectively, taking into account stereochemical considerations as outlined by Lobell and Crout (40), shows that these reaction intermediates fit well into this pocket (Fig. 7). The substrate atoms are surrounded by a number of amino acid side chains, Asp27, His113, His114, Tyr290, Thr388, and Glu473 (conserved residues underlined), which might be involved in substrate binding and catalysis. Based on modelling studies (40) it was suggested that Glu477 of ScPDC (the residue corresponding to Glu473 in ZmPDC) plays a key role in both pyruvate decarboxylation, leading to the intermediate 2-(1-hydroxyethyl) ThDP, and the subsequent protonation of this compound, resulting in the release of the product, acetaldehyde. In the first case Glu477 is thought to stabilize the dianion formed after nucleophilic attack of the thiazolium carbanion on pyruvate through a hydrogen bond, which would require the side chain of Glu477 to be protonated (40). In ZmPDC, in addition to the side chain of Glu473, the hydroxyl group of Tyr290 points toward the carboxyl group of 2-(2-hydroxypropionyl) ThDP and seems sufficiently close to form a hydrogen bond and contribute to the stabilization of the negative charge of the carboxyl group.
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Carboligase Activity-- A side reaction catalyzed by pyruvate decarboxylases is the carboligase activity, where the activated acetaldehyde bound to ThDP is condensed to a second aldehyde molecule. This acetoin-type condensation is of considerable industrial interest, for example for the synthesis of (R)-1-hydroxy-1-phenylpropan-2-one, an intermediate in the synthesis of L-ephidrine. The carboligase activity of PDC from yeast is 20-fold higher compared with the bacterial enzyme (42); however, because of the higher stability of the latter, it would be desirable to increase the carboligase activity of ZmPDC. The differences in activity are most likely because of steric hindrance at the active site in ZmPDC. When the solvent-accessible surface is calculated for all available PDC structures with a model of the key intermediate, 2-(1-hydroxyethyl) ThDP included, an accessible cavity was found at the active site in ScPDC, which could bind the second aldehyde substrate. In ZmPDC, this cavity is filled by bulky amino acid side chains, e.g. Tyr290 and Trp392. The latter has been mutated into an alanine, which results in a 5-fold increase of the carboligase activity in ZmPDC (43). Further improvement for the carboligase activity can be expected by increasing the size of this cavity, for example by replacement of the side chain of Tyr290.
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FOOTNOTES |
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* This work was supported by grants from the Swedish Natural Science Research Council, the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie, and the Deutscher Akademischer Austauschdienst.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and structure factors (code 1ZPD) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
¶ To whom correspondence should be addressed: Dept. of Medical Biochemistry and Biophysics, Doktorsringen 9, Karolinska Institutet, S-17177 Stockholm, Sweden. Tel: 46-8-728-7651; Fax: 46-8-32-7626; E-mail: guoguang{at}alfa.mbb.ki.se.
The abbreviations used are: PDC, pyruvate decarboxylase; ZmPDC, pyruvate decarboxylase from Z. mobilisScPDC, pyruvate decarboxylase from Saccharomyces cerevisiaeThDP, thiamin diphosphateNCS, noncrystallographic symmetryMes, 4-morpholineethanesulfonic acidPOX, pyruvate oxidase.
2 "Form A" and "form B" ScPDC denote yeast pyruvate decarboxylase crystallized in the absence or presence of the allosteric activator, pyruvamide.
3 D. Dobritzsch, S. König, G. Schneider, and G. Lu, unpublished observation.
4 G. Lu, http://gamma.mbb.ki.se/~guoguang/watncs.html.
5 G. Lu, manuscript in preparation.
6 To facilitate comparison, we are using the nomenclature defined by Muller et al. (34) to identify the various domains in ThDP-dependent enzymes.
7 S. König, D. Svergun, and M. H. J. Koch, unpublished results.
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REFERENCES |
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