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• W1998113863 abstract "Thiamine diphosphate (ThDP)-dependent enzymes are ubiquitously present in all organisms and catalyze essential reactions in various metabolic pathways. ThDP-dependent phosphoketolase plays key roles in the central metabolism of heterofermentative bacteria and in the pentose catabolism of various microbes. In particular, bifidobacteria, representatives of beneficial commensal bacteria, have an effective glycolytic pathway called bifid shunt in which 2.5 mol of ATP are produced per glucose. Phosphoketolase catalyzes two steps in the bifid shunt because of its dual-substrate specificity; they are phosphorolytic cleavage of fructose 6-phosphate or xylulose 5-phosphate to produce aldose phosphate, acetyl phosphate, and H2O. The phosphoketolase reaction is different from other well studied ThDP-dependent enzymes because it involves a dehydration step. Although phosphoketolase was discovered more than 50 years ago, its three-dimensional structure remains unclear. In this study we report the crystal structures of xylulose 5-phosphate/fructose 6-phosphate phosphoketolase from Bifidobacterium breve. The structures of the two intermediates before and after dehydration (α,β-dihydroxyethyl ThDP and 2-acetyl-ThDP) and complex with inorganic phosphate give an insight into the mechanism of each step of the enzymatic reaction. Thiamine diphosphate (ThDP)-dependent enzymes are ubiquitously present in all organisms and catalyze essential reactions in various metabolic pathways. ThDP-dependent phosphoketolase plays key roles in the central metabolism of heterofermentative bacteria and in the pentose catabolism of various microbes. In particular, bifidobacteria, representatives of beneficial commensal bacteria, have an effective glycolytic pathway called bifid shunt in which 2.5 mol of ATP are produced per glucose. Phosphoketolase catalyzes two steps in the bifid shunt because of its dual-substrate specificity; they are phosphorolytic cleavage of fructose 6-phosphate or xylulose 5-phosphate to produce aldose phosphate, acetyl phosphate, and H2O. The phosphoketolase reaction is different from other well studied ThDP-dependent enzymes because it involves a dehydration step. Although phosphoketolase was discovered more than 50 years ago, its three-dimensional structure remains unclear. In this study we report the crystal structures of xylulose 5-phosphate/fructose 6-phosphate phosphoketolase from Bifidobacterium breve. The structures of the two intermediates before and after dehydration (α,β-dihydroxyethyl ThDP and 2-acetyl-ThDP) and complex with inorganic phosphate give an insight into the mechanism of each step of the enzymatic reaction. Bifidobacteria represent a ubiquitous commensal bacterial group in the gastrointestinal tract of humans and animals (1Guarner F. Malagelada J.R. Lancet. 2003; 361: 512-519Abstract Full Text Full Text PDF PubMed Scopus (2437) Google Scholar). A unique central hexose fermentation pathway of bifidobacteria is called the “bifid shunt,” which is summarized in the following scheme (2Scardovi V. Trovatelli L.D. Ann. Microbiol. 1965; 15: 19-29Google Scholar).2 glucose +5Pi+5ADP→3 acetate +2 lactate +5ATP+5H2OSCHEME 1 Therefore, ATP production by the bifid shunt is 1.25-fold more effective than that by lactic acid fermentation in the well known Embden-Meyerhof glycolytic pathway (2 ATP per glucose). Two thiamine diphosphate (ThDP) 3The abbreviations used are: ThDPthiamine diphosphateTKtransketolasePKphosphoketolasePOXpyruvate oxidaseacetyl-Pacetyl phosphateXPKxylulose 5-phosphate phosphoketolaseX5Pxylulose 5-phosphateF6Pfructose 6-phosphateXFPKxylulose 5-phosphate/fructose 6-phosphate phosphoketolaseDHEThDPα,β-dihydroxyethyl ThDPAcThDP2-acetyl-ThDPBbXFPKXFPK from B. breveScTKTK from S. cerevisiaeLpPOXPOX from L. plantarumEcTKTK from E. coliBicineN,N-bis(2-hydroxyethyl)glycine.-dependent enzymes, transketolase (TK) and phosphoketolase (PK), catalyze key steps of the bifid shunt (Fig. 1). The structure and mechanism of TK has been extensively studied (3Schneider G. Lindqvist Y. Biochim. Biophys. Acta. 1998; 1385: 387-398Crossref PubMed Scopus (81) Google Scholar, 4Fiedler E. Thorell S. Sandalova T. Golbik R. König S. Schneider G. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 591-595Crossref PubMed Scopus (108) Google Scholar). However, the three-dimensional structure and reaction mechanism of PK has long been enigmatic, although PK was discovered in 1958 (5Heath E.C. Hurwitz J. Horecker B.L. Ginsburg A. J. Biol. Chem. 1958; 231: 1009-1029Abstract Full Text PDF PubMed Google Scholar, 6Hurwitz J. Biochim. Biophys. Acta. 1958; 28: 599-602Crossref PubMed Scopus (25) Google Scholar, 7Schramm M. Klybas V. Racker E. J. Biol. Chem. 1958; 233: 1283-1288Abstract Full Text PDF PubMed Google Scholar). thiamine diphosphate transketolase phosphoketolase pyruvate oxidase acetyl phosphate xylulose 5-phosphate phosphoketolase xylulose 5-phosphate fructose 6-phosphate xylulose 5-phosphate/fructose 6-phosphate phosphoketolase α,β-dihydroxyethyl ThDP 2-acetyl-ThDP XFPK from B. breve TK from S. cerevisiae POX from L. plantarum TK from E. coli N,N-bis(2-hydroxyethyl)glycine. ThDP is a biologically active form of vitamin B1. ThDP-dependent enzymes are ubiquitously present in all organisms and catalyze various essential reactions in metabolic pathways. These enzymes generally catalyze the conversion of 2-keto acid and require ThDP and divalent cations as cofactors. Formation of ThDP ylide, which is accomplished by deprotonation of the C2 atom on the thiazolium ring, is the first essential activation step (Fig. 2) (8Breslow R. J. Am. Chem. Soc. 1958; 80: 3719-3726Crossref Scopus (1414) Google Scholar, 9Kluger R. Tittmann K. Chem. Rev. 2008; 108: 1797-1833Crossref PubMed Scopus (209) Google Scholar). ThDP-dependent enzymes are divided into four families based on their primary and tertiary structures (10Duggleby R.G. Acc. Chem. Res. 2006; 39: 550-557Crossref PubMed Scopus (76) Google Scholar). Three of the four families catalyze oxidative decarboxylation reactions to produce biologically essential metabolites such as acetyl-CoA (9Kluger R. Tittmann K. Chem. Rev. 2008; 108: 1797-1833Crossref PubMed Scopus (209) Google Scholar, 11Jordan F. Nat. Prod. Rep. 2003; 20: 184-201Crossref PubMed Scopus (198) Google Scholar, 12Tittmann K. FEBS J. 2009; 276: 2454-2468Crossref PubMed Scopus (41) Google Scholar). Acetyl phosphate (acetyl-P)-producing pyruvate oxidase (POX), which belongs to one of the oxidative decarboxylation-catalyzing families, cleaves pyruvate in the presence of inorganic phosphate (Pi) and O2 to produce acetyl-P, CO2, and H2O2 (Fig. 2) (13Muller Y.A. Schulz G.E. Science. 1993; 259: 965-967Crossref PubMed Scopus (215) Google Scholar). TK is a representative member of the fourth family (known as the TK family). The reactions of enzymes belonging to this family are significantly different from those belonging to other families because the TK family enzymes catalyze non-oxidative reactions. TK catalyzes the cleavage of a C-C bond in ketose phosphate (donor substrate) into a two-carbon fragment and aldose phosphate. This cleavage is subsequently followed by condensation of the ThDP-bound two-carbon fragment with aldose (acceptor substrate). PK belongs to the TK family, and there are two types of enzymes with different preferences for sugar phosphate substrates exist. Xylulose-5-phosphate PK (XPK, EC 4.1.2.9) prefers xylulose 5-phosphate (X5P) to fructose 6-phosphate (F6P) (5Heath E.C. Hurwitz J. Horecker B.L. Ginsburg A. J. Biol. Chem. 1958; 231: 1009-1029Abstract Full Text PDF PubMed Google Scholar, 6Hurwitz J. Biochim. Biophys. Acta. 1958; 28: 599-602Crossref PubMed Scopus (25) Google Scholar), whereas X5P/F6P PK (XFPK, EC 4.1.2.22) acts on both X5P and F6P with comparable activities (7Schramm M. Klybas V. Racker E. J. Biol. Chem. 1958; 233: 1283-1288Abstract Full Text PDF PubMed Google Scholar, 14Goldberg M.L. Racker E. J. Biol. Chem. 1962; 237: 3841-3842Abstract Full Text PDF PubMed Google Scholar, 15Sgorbati B. Lenaz G. Casalicchio F. Antonie Van Leeuwenhoek. 1976; 42: 49-57Crossref PubMed Scopus (28) Google Scholar, 16Meile L. Rohr L.M. Geissmann T.A. Herensperger M. Teuber M. J. Bacteriol. 2001; 183: 2929-2936Crossref PubMed Scopus (101) Google Scholar). These enzymes constitute two homologous (sequence identity > 40%) but phylogenetically distinct groups (supplemental Figs. 1 and 2 and supplemental Table 1) (17Sánchez B. Zúñiga M. González-Candelas F. de los Reyes-Gavilán C.G. Margolles A. J. Mol. Microbiol. Biotechnol. 2010; 18: 37-51Crossref PubMed Scopus (27) Google Scholar). PKs catalyze the cleavage of X5P or F6P (donor) utilizing Pi as the acceptor (phosphorolysis) to produce acetyl-P, water, and glyceraldehyde 3-phosphate or erythrose 4-phosphate.X5P+Pi→ acetyl-P +H2O+ glyceraldehyde 3 -phosphate (XPK and XFPK) (Eq. 1) F6P+Pi→ acetyl- P+H2O+ erythrose 4 -phosphate (XFPK)(Eq. 2) The XFPK-type gene has only been found in bifidobacteria, and XFPK catalyzes two steps in the bifid shunt because of its dual-substrate specificity (Fig. 1). Because PK activity against F6P is specific to the bifid shunt, it is employed as the most reliable non-molecular test for identification of bifidobacteria (18Scardovi V. Sneath P.H.A. Mair N.S. Sharpe M.E. Holt J.G. Bergey's Manual of Systematic Bacteriology. Williams and Wilkins, Baltimore1986: 1418-1434Google Scholar, 19Vlková E. Nevoral J. Jencikova B. Kopecný J. Godefrooij J. Trojanová I. Rada V. J. Microbiol. Methods. 2005; 60: 365-373Crossref PubMed Scopus (47) Google Scholar). XPK is a key enzyme in the pentose catabolism in various microbes, including filamentous fungi and yeasts (17Sánchez B. Zúñiga M. González-Candelas F. de los Reyes-Gavilán C.G. Margolles A. J. Mol. Microbiol. Biotechnol. 2010; 18: 37-51Crossref PubMed Scopus (27) Google Scholar). This pentose catabolism is called the “PK pathway” (20Panagiotou G. Andersen M.R. Grotkjaer T. Regueira T.B. Hofmann G. Nielsen J. Olsson L. PloS one. 2008; 3: e3847Crossref PubMed Scopus (37) Google Scholar). Furthermore, the PK pathway is the central pathway in the metabolism of heterofermentative lactic acid bacteria including the genera Lactobacillus and Leuconostoc. Heterofermentative lactic acid bacteria, representatives of beneficial gut microbes (probiotics), produce lactic and acetic acids as the main end products. These short-chain fatty acids are important for hosts, not only because they prevent the growth of harmful bacteria by lowering the intestinal pH but also because they serve as an energy source for intestinal epithelial cells. In addition, short-chain fatty acids can modulate intestinal immune and inflammatory responses via G-protein-coupled receptors (21Maslowski K.M. Vieira A.T. Ng A. Kranich J. Sierro F. Yu D. Schilter H.C. Rolph M.S. Mackay F. Artis D. Xavier R.J. Teixeira M.M. Mackay C.R. Nature. 2009; 461: 1282-1286Crossref PubMed Scopus (2133) Google Scholar). Recently, kinetic analysis of PK-2 from Lactobacillus plantarum, belonging to the XPK group, revealed that the reaction follows a ping-pong bi-bi mechanism, leading to the proposal that the first-half of the reaction by PK proceeds through the same mechanism as TK and forms an α,β-dihydroxyethyl ThDP (DHEThDP) intermediate (Fig. 2) (22Yevenes A. Frey P.A. Bioorg Chem. 2008; 36: 121-127Crossref PubMed Scopus (23) Google Scholar). However, the subsequent reaction catalyzed by PK is distinct from TK at the following two points; 1) the dehydration reaction occurs, and 2) the acceptor substrate Pi is believed to attack the possible 2-acetyl-ThDP (AcThDP) intermediate, similar to the case of acetyl-P-producing POX. Recently, preliminary x-ray crystallographic studies of XPK from Lactococcus lactis (23Petrareanu G. Balasu M.C. Zander U. Scheidig A.J. Szedlacsek S.E. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2010; 66: 805-807Crossref PubMed Scopus (5) Google Scholar) and XFPK from Bifidobacterium breve 203 (BbXFPK) (24Suzuki R. Kim B.-J. Iwamoto Y. Katayama T. Ashida H. Wakagi T. Shoun H. Fushinobu S. Yamamoto K. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2010; 66: 941-943Crossref PubMed Scopus (8) Google Scholar) are reported. In this study we report the crystal structure of BbXFPK as the first three-dimensional structure of PK. The structures of the resting form, the two intermediates before and after dehydration (DHEThDP and AcThDP), the complex with the acceptor substrate Pi, and four mutant enzymes have been determined. This study revealed a structural basis for the reaction mechanism of a ThDP-dependent enzyme involving dehydration and nucleophilic attack of Pi to the AcThDP intermediate. Construction of the expression vector, protein expression, purification, and kinetic analysis employing F6P as the donor substrate were performed as described previously (24Suzuki R. Kim B.-J. Iwamoto Y. Katayama T. Ashida H. Wakagi T. Shoun H. Fushinobu S. Yamamoto K. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2010; 66: 941-943Crossref PubMed Scopus (8) Google Scholar). Mutants of BbXFPK were constructed using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) using the primers summarized in supplemental Table 2. Native (resting) BbXFPK and its mutants were crystallized according to the method described previously (24Suzuki R. Kim B.-J. Iwamoto Y. Katayama T. Ashida H. Wakagi T. Shoun H. Fushinobu S. Yamamoto K. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2010; 66: 941-943Crossref PubMed Scopus (8) Google Scholar). Structures of AcThDP intermediate (BbXFPK/AcThDP), DHEThDP intermediate (BbXFPK/DHEThDP), and BbXFPK in complex with the acceptor substrate Pi (BbXFPK/Pi) were trapped using cryocrystallography technique. Crystals of BbXFPK/AcThDP were prepared by soaking native BbXFPK crystals in mother liquor (24% (v/v) PEG 6000, 0.1 m Bicine buffer, pH 9.0) containing 27 mm F6P for various time periods at room temperature. BbXFPK/DHEThDP crystals were obtained by soaking in 54 mm F6P solution for 15 s. Crystals of BbXFPK/Pi were obtained by cocrystallization with 5 mm potassium phosphate. The mother liquor containing 20% ethylene glycol was used as a cryoprotectant, except for the BbXFPK/Pi complex for which 20% glycerol was used. Selenomethionine-labeled enzyme was expressed in Escherichia coli B834 (DE3) (Novagen, Madison, WI). Diffraction data were collected using beamlines at SPring-8 (Harima, Japan) and Photon Factory (Tsukuba, Japan). Diffraction data were processed using HKL2000 (25Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38517) Google Scholar). Initial phases were calculated using SnB (26Rappleye J. Innus M. Weeks C.M. Miller R. J. Appl. Crystallogr. 2002; 35: 374-376Crossref Scopus (37) Google Scholar) and Solve/Resolve (27Terwilliger T.C. Berendzen J. Acta. Crystallogr. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3219) Google Scholar). Initial model building was performed using ARP/wARP (28Perrakis A. Morris R. Lamzin V.S. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2563) Google Scholar). Manual model rebuilding and refinement was achieved using Coot (29Emsley P. Cowtan K. Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23207) Google Scholar) and REFMAC5 (30Murshudov G.N. Vagin A.A. Dodson E.J. Acta. Crystallogr. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13850) Google Scholar). Parameters for refinement were generated by the PRODRG server (31Schüttelkopf A.W. van Aalten D.M. Acta. Crystallogr. D Biol. Crystallogr. 2004; 60: 1355-1363Crossref PubMed Scopus (4246) Google Scholar). Details of data collection and refinement statistics are given in Table 1 and supplemental Table 3. Figures were prepared using PyMol (DeLano Scientific, Palo Alto, CA).TABLE 1Data collection and refinement statisticsData setNative restingNative/AcThDPNative/DHEThDPNative/PiSAD peakData collection statistics Space groupI422 Unit cell (Å)a = 174.8a = 174.4a = 173.5a = 173.9a = 174.0b = 174.8b = 174.4b = 173.5b = 173.9b = 174.0c = 163.8c = 163.8c = 163.5c = 163.5c = 163.8 Beam linePFAR-NE3APF-BL5APFAR-NW12APF-BL6ASpring-8 BL38B1 Wavelength (Å)1.000001.000001.000000.978000.97875 Resolution (Å)aValues for highest resolution shell are given in parentheses.50-1.70 (1.73-1.70)50-1.90 (1.93-1.90)50-2.10 (2.14-2.10)50-2.30 (2.38-2.30)50-2.60 (2.69-2.60) Total reflections2,049,6891,442,1051,079,159633,806575,648 Unique reflections137,59098,94072,41755,66938,884 Completeness (%)aValues for highest resolution shell are given in parentheses.100 (100)99.9 (100)100 (100)100 (100)99.9 (100) Rmerge (%)aValues for highest resolution shell are given in parentheses.6.5 (32.2)7.0 (34.9)7.1 (29.8)10.8 (35.5)6.7 (22.1) I/σIaValues for highest resolution shell are given in parentheses.49.6 (7.4)52.6 (9.0)42.8 (11.0)24.9 (4.7)32.9 (9.4) RedundancyaValues for highest resolution shell are given in parentheses.14.9 (14.8)14.6 (14.7)14.9 (15.0)11.4 (10.4)7.8 (7.7)Refinement statistics Resolution range (Å)34.18-1.7034.21-1.9049.07-2.1040.88-2.30 No. of reflections130,62793,68168,52452,685 R-factor/Rfree (%)bRfree actor was calculated using 5% of the unique reflections.15.0/18.116.2/19.716.1/20.617.7/22.6 r.m.s.d. from idealBond lengths (Å)0.0370.0300.0280.023Bond angles (°)2.7222.2122.0661.904 Average B-factor (Å2)Protein18.122.720.928.1Water30.233.432.132.6ThDP16.518.919.120.9Mg2+13.216.115.421.2 Ramachandran plot (%)cCalculated by RAMPAGE (45).Favored96.696.696.195.5Allowed3.43.43.94.4Outlier0.00.00.00.1a Values for highest resolution shell are given in parentheses.b Rfree actor was calculated using 5% of the unique reflections.c Calculated by RAMPAGE (45Lovell S.C. Davis I.W. Arendall 3rd, W.B. de Bakker P.I. Word J.M. Prisant M.G. Richardson J.S. Richardson D.C. Proteins. 2003; 50: 437-450Crossref PubMed Scopus (3844) Google Scholar). Open table in a new tab The crystal structures (residues 5–806) of the resting form, the AcThDP intermediate, the DHEThDP intermediate, and the complex with Pi were determined at 1.7, 1.9, 2.1, and 2.3 Å, respectively (Table 1). The dimeric and monomeric structures of BbXFPK are shown in Fig. 3, A and B. BbXFPK consists of three α/β-fold domains; N-terminal (PP domain, residues 5–378), middle (Pyr domain, residues 379–611), and C-terminal (residues 612–806) domains. The crystals contain one molecule per asymmetric unit, and a crystallographic 2-fold axis is consistent with the molecular axis of the dimer. The active site is positioned at the interface between the PP and Pyr domains from different subunits to form a deep and narrow substrate channel, and the reactive C2 atom of ThDP is only accessible from the solvent. These observations suggest that the minimum functional unit of the enzyme is a homodimer. The overall architectures of the monomer and the tightly packed homodimer of BbXFPK are basically similar to those of TK (Fig. 3C) (3Schneider G. Lindqvist Y. Biochim. Biophys. Acta. 1998; 1385: 387-398Crossref PubMed Scopus (81) Google Scholar), with a root mean square deviation of 2.6 Å for 545 α-carbon atoms. However, they share very low sequence homology, with a sequence identity of 15% based on structural alignment with TK from Saccharomyces cerevisiae (ScTK). Three active peaks of BbXFPK appeared on gel filtration chromatography with different estimated sizes (24Suzuki R. Kim B.-J. Iwamoto Y. Katayama T. Ashida H. Wakagi T. Shoun H. Fushinobu S. Yamamoto K. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2010; 66: 941-943Crossref PubMed Scopus (8) Google Scholar). A sample from the major homohexameric peak successfully crystallized, whereas those from the two minor peaks (homodimer and homotetramer) failed to crystallize. We examined the crystal packing, but there was no strong interaction that possibly interconnects the dimer units (data not shown). The biological assembly might have collapsed under the crystallization condition. In all structures determined here, ThDP binds in a typical V-conformation (Fig. 4). The pyrimidine and thiazolium rings are held mainly through the hydrophobic residues of the PP and Pyr domains (supplemental Fig. 3A). The diphosphate group of ThDP and the hexacoordinated Mg2+ are anchored to the PP domain through many interactions (supplemental Fig. 3B). Several histidine residues in the active site as well as other residues important for catalysis are completely conserved between BbXFPK and TK (discussed later). In the initial reaction step of all ThDP-dependent enzymes, formation of reactive ThDP ylide is achieved by cofactor-assisted deprotonation of the C2 atom of the thiazolium ring by the N4′-imino group of the pyrimidine ring, and the imino tautomer is stabilized by a completely conserved Glu residue (9Kluger R. Tittmann K. Chem. Rev. 2008; 108: 1797-1833Crossref PubMed Scopus (209) Google Scholar, 32Kern D. Kern G. Neef H. Tittmann K. Killenberg-Jabs M. Wikner C. Schneider G. Hübner G. Science. 1997; 275: 67-70Crossref PubMed Scopus (239) Google Scholar). In the resting structure, interactions between the N4′ atom and the reactive C2 atom (2.9 Å) and between the N1′ atom of pyrimidine and the conserved Glu479 (2.7 Å) residue are present (Fig. 4A). Replacement of this residue (E479A) completely abolished the activity (Table 2). These results indicate that the initial activation mechanism of BbXFPK to form ThDP ylide is identical to that of other ThDP-dependent enzymes.TABLE 2Kinetic parameters for wild-type and mutant BbXFPKEnzymeKcatApparent KmCofactor in mutant structuresaCrystallographic data collected after soaking in 27 mm F6P for 5 min. See supplemental Table 3 for details.F6PPimin−1mmWTbThe data are taken from (24).1,540 ± 609.7 ± 0.31.2 ± 0.2H64ANAcNA, no activity was detected.NANATricyclic ring form of ThDPH64NNANANAH97ANANANA(CDNGdCDNG, crystals did not grow.)H97NNANANA(CDNGdCDNG, crystals did not grow.)H142A12.2 ± 0.97.4 ± 0.61.7 ± 0.2AcThDPH142N39.2 ± 1.27.2 ± 0.82.6 ± 0.5H320ANANANAAcThDPH320NNANANAQ321A59.5 ± 2.54.4 ± 0.31.6 ± 0.2S440A350 ± 1153.5 ± 1.80.57 ± 0.04E479ANANANAY501F197 ± 41.4 ± 0.225.5 ± 1.7H548A100 ± 32.3 ± 0.70.52 ± 0.16N549ANANANAH553ANANANAThDPH553NNANANAK605ANANANAa Crystallographic data collected after soaking in 27 mm F6P for 5 min. See supplemental Table 3 for details.b The data are taken from (24Suzuki R. Kim B.-J. Iwamoto Y. Katayama T. Ashida H. Wakagi T. Shoun H. Fushinobu S. Yamamoto K. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2010; 66: 941-943Crossref PubMed Scopus (8) Google Scholar).c NA, no activity was detected.d CDNG, crystals did not grow. Open table in a new tab When we collected diffraction data from crystals soaked in 27 mm F6P for 5 min in the absence of Pi, extra electron density was observed (Fig. 4B). The density corresponds to three branched carbon or oxygen atoms and is covalently attached to the C2 atom of the thiazolium ring. The appearance of the electron density did not change when the soaking time was varied in a range from 1 to 60 min (data not shown). The central Cα atom appears to be sp2-hybridized because the map of the covalent adduct is trigonal planar (Fig. 4B, inset). Based on these observations, we concluded that the covalent adduct is an acetyl group. It has been proposed that AcThDP formed after dehydration is a stable intermediate of PK in the absence of the acceptor, Pi (22Yevenes A. Frey P.A. Bioorg Chem. 2008; 36: 121-127Crossref PubMed Scopus (23) Google Scholar, 33Frey P.A. Biofactors. 1989; 2: 1-9PubMed Google Scholar). One of the two terminal atoms is located close to the NE2 atom of His-553 (2.5 Å) and the N4′ atom of the pyrimidine ring (3.2 Å) (Fig. 5A, gray). Superimposition with the ScTK-DHEThDP structure illustrates that this atom overlaps with the Cα hydroxyl group of DHEThDP (Fig. 5B), suggesting that it is an oxygen atom. Careful inspection of the heights of electron density peaks and temperature factors supported this assignment of terminal oxygen and carbon atoms. We refined the structure using the parameters of keto-AcThDP. The resolution was not sufficiently high to determine whether it is a keto or an enol form. The planar character of the Cα atom is consistent with the similar AcThDP adduct of L. plantarum POX (LpPOX) (34Wille G. Meyer D. Steinmetz A. Hinze E. Golbik R. Tittmann K. Nat. Chem. Biol. 2006; 2: 324-328Crossref PubMed Scopus (98) Google Scholar). A water molecule is located 3.2 Å from the terminal Cβ atom of the acetyl group (discussed later). Then we prepared a crystal by soaking in 54 mm F6P for a short time period (15 s), and a clear electron density peak corresponding to the Cβ hydroxyl group of DHEThDP was observed (Fig. 4C). The electron density map for the DHE moiety is relatively weak but is sufficiently clear in shape, and is interpreted as DHEThDP. An electron density peak that corresponds to the water molecule in the AcThDP structure was also observed, but the distance from the Cβ hydroxyl atom (Oβ) was abnormally close (2.0 Å). We repeatedly refined the crystal structure by changing the occupancies of these elements (the DHE moiety and the water molecule) and then examined the resultant |Fo| − |Fc| difference map. We finished the refinement by setting the occupancies to 0.7 for the water molecule (B factor = 9.8 Å2), 0.3 for the Oβ atom (B factor = 16.3 Å2), and 0.5 for the other three atoms of the DHE moiety (Cα, Oα, and Cβ; average B factor = 24.9 Å2). The average B factor of the remaining moiety of the ThDP cofactor was 18.8 Å2. Thus, this crystal was assumed to be a mixture of free ThDP with the water (0.5 fraction), DHEThDP without the water (0.3), and AcThDP with the water (0.2). This result indicates that the DHEThDP intermediate is transient under this condition. The map around the central Cα atom is not planar, suggesting that this atom has an sp3-hybridized character (Fig. 4C, inset). Because of limited resolution (2.1 Å), low occupancy (0.3), and the electron density overlaps with those of other states, the hybridization state of the Cα atom cannot be determined unambiguously. However, crystallographic refinement with parameters of the sp2 hybridized (flat) Cα atom resulted in the appearance of significant difference peaks near the Oα and Oβ atoms in the |Fo| − |Fc| map (data not shown). Observation of the sp3-hybridized Cα atom suggests that the resonance hybrid between the neutral enamine and zwitterionic α-carbanion in our DHEThDP structure is unlikely present. Instead, the structure is likely a protonated state at the Cα atom with the S-configuration (S-α,β-dihydroxyethyl-ThDP; Fig. 2, boxed with a broken line). Although the crystal structure was obtained at pH 9.0, the local environment may stabilize the protonated state. In contrast, the DHEThDP intermediate of ScTK was observed as an sp2-hybridized enamine character (Fig. 5C) (4Fiedler E. Thorell S. Sandalova T. Golbik R. König S. Schneider G. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 591-595Crossref PubMed Scopus (108) Google Scholar). The terminal Cβ hydroxyl group interacts with the NE2 atom of His-142 (2.9 Å), the main chain oxygen atom of Gly-155 (2.9 Å), and the N4′ group of the pyrimidine moiety (3.2 Å). The branched Cα hydroxyl group interacts with the NE2 atom of His-553 (2.6 Å), but the N4′ atom of pyrimidine moiety is distant (3.6 Å). The fourth structure, a complex with the acceptor substrate, Pi, was prepared by cocrystallization in the presence of Pi. A clear tetrahedral electron density peak was observed near the C2 atom of the thiazolium ring (Fig. 4D). The four structures of the wild-type BbXFPK determined in this work did not show any significant conformational changes around the active site. This observation indicates that the formation of reaction intermediates and binding of the acceptor substrate do not induce remarkable conformational change, which is consistent with previous findings in ScTK (4Fiedler E. Thorell S. Sandalova T. Golbik R. König S. Schneider G. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 591-595Crossref PubMed Scopus (108) Google Scholar) and LpPOX (34Wille G. Meyer D. Steinmetz A. Hinze E. Golbik R. Tittmann K. Nat. Chem. Biol. 2006; 2: 324-328Crossref PubMed Scopus (98) Google Scholar). The substrate binding channel of ScTK has been identified by the complex structure with erythrose 4-phosphate (35Nilsson U. Meshalkina L. Lindqvist Y. Schneider G. J. Biol. Chem. 1997; 272: 1864-1869Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Moreover, the crystal structures of TK from E. coli (EcTK) in complex with uncleaved donor substrates, tetrahedral covalent X5P-ThDP and F6P-ThDP adducts, have been reported (36Asztalos P. Parthier C. Golbik R. Kleinschmidt M. Hübner G. Weiss M.S. Friedemann R. Wille G. Tittmann K. Biochemistry. 2007; 46: 12037-12052Crossref PubMed Scopus (81) Google Scholar). In the tetrahedral substrate-cofactor adducts, the C2-Cα bond between the cofactor and substrate is distorted from the planarity, and the leaving group is perpendicularly orientated to the thiazolium ring of ThDP (Fig. 6). The out-of-plane distortions of the C2-Cα bond and the perpendicular orientation of the leaving groups are considered to facilitate the elimination of the first product (9Kluger R. Tittmann K. Chem. Rev. 2008; 108: 1797-1833Crossref PubMed Scopus (209) Google Scholar, 37Tittmann K. Wille G. J. Mol. Catal. B Enzym. 2009; 61: 93-99Crossref Scopus (16) Google Scholar). Therefore, the covalent F6P-ThDP adduct of BbXFPK is also expected to adopt a similar conformation. Superimposition of the resting BbXFPK and F6P-ThDP adduct of EcTK revealed that F6P can bind in the active site channel of BbXFPK without any steric hindrance, leading to the estimation of residues involved in substrate recog" @default.
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