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W2125576777 abstract "The crystal structure of tetrameric pyruvate decarboxylase from Zymomonas mobilis has been determined at 1.9 Å resolution and refined to a crystallographicR-factor of 16.2% and Rfree of 19.7%. The subunit consists of three domains, all of the α/β 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. The crystal structure of tetrameric pyruvate decarboxylase from Zymomonas mobilis has been determined at 1.9 Å resolution and refined to a crystallographicR-factor of 16.2% and Rfree of 19.7%. The subunit consists of three domains, all of the α/β 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. Pyruvate decarboxylase (PDC) 1The abbreviations used are: PDCpyruvate decarboxylaseZmPDCpyruvate decarboxylase from Z. mobilisScPDCpyruvate decarboxylase from Saccharomyces cerevisiaeThDPthiamin diphosphateNCSnoncrystallographic symmetryMes4-morpholineethanesulfonic acidPOXpyruvate oxidase.1The abbreviations used are: PDCpyruvate decarboxylaseZmPDCpyruvate decarboxylase from Z. mobilisScPDCpyruvate decarboxylase from Saccharomyces cerevisiaeThDPthiamin diphosphateNCSnoncrystallographic symmetryMes4-morpholineethanesulfonic acidPOXpyruvate oxidase. 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 (1Bringer-Meyer S. Schimz K.L. Sahm H. Arch. Microbiol. 1986; 146: 105-110Crossref Scopus (76) Google Scholar). In this organism, PDC amounts to 4% of the total soluble protein and to 10% of the extractable protein after cell lysis (1Bringer-Meyer S. Schimz K.L. Sahm H. Arch. Microbiol. 1986; 146: 105-110Crossref Scopus (76) Google Scholar). There appears to be only one gene coding for this enzyme in Z. mobilis (2Neale A.D. Scopes R.K. Wettenhall R.E.H. Hoogenraad N.J. J. Bacteriol. 1987; 169: 1024-1028Crossref PubMed Scopus (72) Google Scholar). pyruvate decarboxylase pyruvate decarboxylase from Z. mobilis pyruvate decarboxylase from Saccharomyces cerevisiae thiamin diphosphate noncrystallographic symmetry 4-morpholineethanesulfonic acid pyruvate oxidase. pyruvate decarboxylase pyruvate decarboxylase from Z. mobilis pyruvate decarboxylase from Saccharomyces cerevisiae thiamin diphosphate noncrystallographic symmetry 4-morpholineethanesulfonic acid pyruvate oxidase. 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 (1Bringer-Meyer S. Schimz K.L. Sahm H. Arch. Microbiol. 1986; 146: 105-110Crossref Scopus (76) Google Scholar, 2Neale A.D. Scopes R.K. Wettenhall R.E.H. Hoogenraad N.J. J. Bacteriol. 1987; 169: 1024-1028Crossref PubMed Scopus (72) Google Scholar). 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 (1Bringer-Meyer S. Schimz K.L. Sahm H. Arch. Microbiol. 1986; 146: 105-110Crossref Scopus (76) Google Scholar, 3Diefenbach R.J. Duggleby R.G. Biochem. J. 1991; 276: 439-445Crossref PubMed Scopus (46) Google Scholar). 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 (4Pohl M. Mesch K. Rodenbrock A. Kula M.-R. Biotechnol. Appl. Biochem. 1995; 22: 95-105Google Scholar). 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 (5Hübner G. Weidhase R. Schellenberger A. Eur. J. Biochem. 1978; 92: 175-181Crossref PubMed Scopus (108) Google Scholar, 6Dietrich A. König S. FEBS Lett. 1997; 400: 42-44Crossref PubMed Scopus (15) Google Scholar). Crystallographic studies of yeast PDC revealed considerable differences in the tetramer assembly between enzyme species obtained in the absence (7Dyda F. Furey W. Swaminathan S. Sax M. Farrenkopf B. Jordan F. Biochemistry. 1993; 32: 6165-6170Crossref PubMed Scopus (226) Google Scholar, 8Arjunan P. Umland T. Dyda F. Swaminathan S. Furey W. Sax M. Farrenkopf B. Gao Y. Zhang D. Jordan F. J. Mol. Biol. 1986; 355: 590-600Google Scholar) or presence of the activator pyruvamide (9Lu G. Dobritzsch D. König S. Schneider G. FEBS Lett. 1997; 403: 249-253Crossref PubMed Scopus (40) Google Scholar). Cross-linking and small angle x-ray solution scattering experiments also indicated significant tetramer reassembly and conformational changes during substrate activation in yeast PDC (10König S. Svergun D. Koch M.H.J. Hübner G. Schellenberger A. Eur. Biophys. J. 1993; 22: 185-194Crossref PubMed Scopus (40) Google Scholar, 11Hübner G. König S. Schellenberger A. Koch M. FEBS Lett. 1990; 266: 17-20Crossref PubMed Scopus (30) Google Scholar, 12König S. Hübner G. Schellenberger A. Biomed. Biochim. Acta. 1990; 49: 465-471PubMed Google Scholar). 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. 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 β-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. 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. 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 (13Otwinowski Z. Proceedings of the CCP4 Study Weekend. Daresbury Laboratory, SERC, Warrington, UK1993: 56-62Google Scholar). 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, α = 103.7 °, β = 94.5 °, γ = 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 TableI.Table IStatistics of data collectionResolution1.86 Å(1.92–1.86 Å)Measured reflections306481(25442)Unique reflections157288(13826)Completeness88.2%51.7%Rmerge0.063(0.210)The values in parentheses are for the highest resolution interval. Open table in a new tab The values in parentheses are for the highest resolution interval. The structure of ZmPDC was determined by molecular replacement using a model of form B 2“Form A” and “form B” ScPDC denote yeast pyruvate decarboxylase crystallized in the absence or presence of the allosteric activator, pyruvamide. ScPDC refined at 2.4 Å (9Lu G. Dobritzsch D. König S. Schneider G. FEBS Lett. 1997; 403: 249-253Crossref PubMed Scopus (40) Google Scholar), 3D. Dobritzsch, S. König, G. Schneider, and G. Lu, unpublished observation. which shares only 28% sequence identity with ZmPDC. Orientation and positions of the molecules were determined using the AMORE program (14Navaza J. Acta Crystallogr. Sec. D. 1992; 50: 157-163Crossref Scopus (5028) Google Scholar) 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. A test set of 4% of the reflections was excluded before starting any crystallographic refinement to monitor the Rfreevalue. Rigid body refinement was carried out with XPLOR (15Brunger A. Kuriyan J. Karplus M. Science. 1987; 235: 458-460Crossref PubMed Scopus (2126) Google Scholar) 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 (16Cowtan K.D. Main P. Acta Crystallogr. Sec. D. 1996; 52: 43-48Crossref PubMed Scopus (288) Google Scholar) in the CCP4 package (17Collaborative Computational Project, Number 4 Acta Crystallogr. Sec. D. 1994; 50: 760-763Crossref PubMed Scopus (19748) Google Scholar) and the Rave packages (18Jones T.A. Dodson E.J. Gover S. Wolf W. Proceedings of the CCP4 Study Weekend. Daresbury Laboratory, Warrington, UK1992: 99-105Google Scholar). Based on the averaged electron density maps, the model was rebuilt using the O program (19Jones T.A. Zou J.-Y. Cowan S. Kjeldgaard M. Acta Crystallogr. Sec. A. 1991; 47: 100-119Google Scholar), 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 (20Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sec. D. 1997; 53: 240-255Crossref PubMed Scopus (13853) Google Scholar), 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 theRfree 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 (17Collaborative Computational Project, Number 4 Acta Crystallogr. Sec. D. 1994; 50: 760-763Crossref PubMed Scopus (19748) Google Scholar), the PEAKCHECK program (written by J. Smith), and the WATNCS program. 4G. Lu,http://gamma.mbb.ki.se/∼guoguang/watncs.html. 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 fromFo − Fc maps by the DIFLIST program 5G. Lu, manuscript in preparation. 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 TableII.Table IIStatistics of structure refinement and the final modelData used for the refinement (Å)15–1.86Number of reflections157061Number of nonhydrogen atoms19877R value0.162Rfree0.197r.m.s. bond lengths (Å)0.007r.m.s. bond angle (°)1.2Bmean of all atoms (Å2)14.9 Open table in a new tab The quality of the model was examined using PROCHECK (21Laskowski R.A. McArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 282-291Crossref Google Scholar). Structural comparisons of ZmPDC with form A ScPDC (PDB accession code 1PVD) (8Arjunan P. Umland T. Dyda F. Swaminathan S. Furey W. Sax M. Farrenkopf B. Gao Y. Zhang D. Jordan F. J. Mol. Biol. 1986; 355: 590-600Google Scholar), form B ScPDC (PDB accession code 1YPD) (9Lu G. Dobritzsch D. König S. Schneider G. FEBS Lett. 1997; 403: 249-253Crossref PubMed Scopus (40) Google Scholar), and pyruvate oxidase (PDB accession code 1POX) (22Muller Y.A. Schulz G.E. Science. 1993; 259: 965-967Crossref PubMed Scopus (215) Google Scholar) were carried out using the TOP program (23Lu G. Protein Data Bank Quarterly Newsletter. 1996; 78: 10-11Google Scholar). The solvent-accessible surface was analyzed with the VOIDOO (24Klejwegt G. Jones T.A. Acta Crystallogr. Sec. D. 1994; 50: 178-185Crossref PubMed Scopus (982) Google Scholar) and the AREAIMOL (17Collaborative Computational Project, Number 4 Acta Crystallogr. Sec. D. 1994; 50: 760-763Crossref PubMed Scopus (19748) Google Scholar) programs. Enzymatic activity was assayed at 30 °C and 340 nm with a NADH/ADH coupled test in 100 mmsodium citrate, pH 6.1, according to Holzer et al. (25Holzer H. Schultz G. Villar-Palasi C. Jüntgen-Sell J. Biochem. Z. 1956; 327: 331-344PubMed Google Scholar). 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 (ε = 275 320m−1 cm−1). 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 Asp530in 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. In total, four amino acid sequences of ZmPDC have been published (26Reynen M. Sahm H. J. Bacteriol. 1988; 170: 3310-3313Crossref PubMed Google Scholar, 27Conway T. Osman Y.A. Konnan J.I. Hoffman E.M. Ingram L.O. J. Bacteriol. 1987; 169: 949-954Crossref PubMed Google Scholar, 28Neale A.D. Scopes R.K. Wettenhall R.E.H. Hoogenraad N.J. Nucleic Acids Res. 1987; 15: 1753-1761Crossref PubMed Scopus (34) Google Scholar, 29Miczka G. Vernau J. Kula M.-R. Hofmann B. Schomburg D. Biotechnol. Appl. Biochem. 1992; 15: 192-206PubMed Google Scholar). Although only one ZmPDC gene has been identified so far (2Neale A.D. Scopes R.K. Wettenhall R.E.H. Hoogenraad N.J. J. Bacteriol. 1987; 169: 1024-1028Crossref PubMed Scopus (72) Google Scholar), all these sequences differ from each other at a few positions. None of these sequences completely matched the electron density map, and the amino acid sequence of our final model (listed in Fig. 4 B) is slightly different from all the templates. The amino acid sequence determined by protein sequencing (29Miczka G. Vernau J. Kula M.-R. Hofmann B. Schomburg D. Biotechnol. Appl. Biochem. 1992; 15: 192-206PubMed Google Scholar) required the fewest changes to be consistent with the electron density map (Glu200 → Asp, Asn256 → Leu, and Ala341 → Ser). In the present model, 92.1% of the amino acid residues are located in the most favored regions of the Ramachandran plot. One residue, Ser74, is located in the disallowed region in all four subunits. This residue is, however, well defined in its electron density. 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, 6To facilitate comparison, we are using the nomenclature defined by Muller et al. (34Muller Y. Lindqvist Y. Furey W. Schulz G.E. Jordan F. Schneider G. Structure. 1993; 1: 95-103Abstract Full Text PDF PubMed Scopus (174) Google Scholar) to identify the various domains in ThDP-dependent enzymes. each with an open α/β topology (Fig. 2). The nomenclature of the secondary structural elements is shown in Figs. 2and 4 B. 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. Two of the monomers are tightly bound to each other and are related by 2-fold symmetry. The extensive interface in the dimer comprises about 4400 Å2, corresponding to 19.4% of the surface area of the monomer. Most of the amino acid residues building up this interface are from the PYR and PP domains, but residues 286–290 of the R domain also are part of this region. In total, 7 pairs of salt bridges, 66 pairs of hydrogen bonds, and extensive hydrophobic interactions are formed between the two subunits. Two such dimers further form a tetramer with pseudo 222 symmetry, with an interface area of 4400 Å2 (12.4% of the dimer surface). All three domains of each subunit are involved in these interfaces. The interactions between the two dimers include 25 sets of salt bridges and 64 sets of hydrogen bonds. At the dimer-dimer interface some narrow but extensive cavities are formed, part of which are occupied by a large number of ordered water molecules. The interactions between the two dimers are not as tight as at the monomer-monomer interface so that the ZmPDC tetramer can be described as a dimer of dimers, similar to ScPDC (7Dyda F. Furey W. Swaminathan S. Sax M. Farrenkopf B. Jordan F. Biochemistry. 1993; 32: 6165-6170Crossref PubMed Scopus (226) Google Scholar, 30König S. Svergun D. Koch M.H.J. Hübner G. Schellenberger A. Biochemistry. 1992; 31: 8726-8731Crossref PubMed Scopus (52) Google Scholar). 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 (7Dyda F. Furey W. Swaminathan S. Sax M. Farrenkopf B. Jordan F. Biochemistry. 1993; 32: 6165-6170Crossref PubMed Scopus (226) Google Scholar), transketolase (31Lindqvist Y. Schneider G. Ermler U. Sundström M. EMBO J. 1992; 11: 2373-2379Crossref PubMed Scopus (304) Google Scholar), and POX (22Muller Y.A. Schulz G.E. Science. 1993; 259: 965-967Crossref PubMed Scopus (215) Google Scholar). 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). During the refinement, it became clear that the ThDP molecule bound to the ZmPDC crystals was a chemically modified analogue of ThDP rather than the cofactor itself. The electron density maps showed no electron density between the assumed position of the C2 carbon atom of the thiazolium ring and the neighboring sulfur and nitrogen atoms, indicating that the bond between these atoms was broken (Fig. 1,bottom). In addition, negative difference electron density was observed at the position of the C2 carbon atom, indicating that the thiazolium ring had been opened and that the C2 carbon atom had been lost. Positive difference electron density was found close to the 4′ amino group of the pyrimidine ring, which has been interpreted as a water molecule tightly bound to the amino group via a hydrogen bond. The well defined electron density for the remaining parts of ThDP, the diphosphate and pyrimidine moieties, does not indicate any significant further chemical modification or degradation (Fig. 1,bottom) and suggests that the bound species might beN-(2-methyl-4-amino-5-pyrimidyl)-N-(1-methyl-2-thiol but(1)en-4-diphosphatidyl) amide. Analysis by mass spectrometry of the ThDP analogue isolated by HPLC from redissolved crystals revealed a mass difference consistent with the loss of a methine carbon atom. The apparent selectivity in binding the acyclic analogue of ThDP most likely reflects the inability of cofactor exchange in the crystals rather than preferential binding. In the crystals, ThDP is buried deeply in its binding site, and cofactor release would require large conformational changes, which are prevented by the crystal packing. Analysis of redissolved crystals of ZmPDC showed that the catalytic activity had been lost. Only when redissolved enzyme was incubated with fresh ThDP under conditions where cofactor exchange can take place could we restore about 55% of the original activity. The modification of bound ThDP is not induced by x-ray radiation during data collection, because loss of catalytic activity is also observed in crystals not exposed to x-ray radiation. It thus appears that during crystallization of ZmPDC, a partial degradation of ThDP occurs leading to an inactive cofactor analogue. The tendency to undergo hydrolytic cleavage of the thiazolium ring is an established feature of thiamin and related compounds (32Zoltewicz J.A. Uray G. J. Org. Chem. 1980; 45: 2104-2108Crossref Scopus (34) Google Scholar). For instance, ring opening can proceed through the formation of a pseudobase by nucleophilic attack of hydroxide on the C2 carbon atom (33Duclos J.M. Haake P. Biochemistry. 1974; 13: 5358-5362Crossref PubMed Scopus (53) Google Scholar). In ZmPDC crystals, the initial nucleophilic attack on the C2 carbon could be delivered by the water molecule that is in hydrogen bonding distance to the 4-amino group. The nucleophilicity of this water molecule could be increased through interactions with the neighboring side chain of His114, which probably is uncharged (see below) and can act as a proton abstractor. The chemical steps that lead to the loss of the C2 carbon atom after ring opening are less clear and require further study. In view of the observed loss of activity in redissolved crystals (restored by the addition of fresh ThDP) and the compelling evidence that thiamin catalysis proceeds though the cyclic thiazolium ion and not an ring-opened form of thiamin (see for example Ref. 33Duclos J.M. Haake P. Biochemistry. 1974; 13: 5358-5362Crossref PubMed Scopus (53) Google Scholar), we consider the acyclic analogue of ThDP at the active site an artifact and not a species of mechanistic relevance. 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. 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 α-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 β-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.Table IIIComparison of domain-domain, subunit-subunit relations and interfaces in pyruvate, decarboxylases, and pyruvate oxidaseMode of superpositionZmPDCform A ScPDCform B ScPDCPOXDomai" @default.
W2125576777 created "2016-06-24" @default.
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W2125576777 creator A5048623337 @default.
W2125576777 creator A5067086667 @default.
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W2125576777 date "1998-08-01" @default.
W2125576777 modified "2023-09-26" @default.
W2125576777 title "High Resolution Crystal Structure of Pyruvate Decarboxylase from Zymomonas mobilis" @default.
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