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• W2003013017 abstract "Thiamine diphosphate-dependent enzymes are involved in a wide variety of metabolic pathways. The molecular mechanism behind active site communication and substrate activation, observed in some of these enzymes, has since long been an area of debate. Here, we report the crystal structures of a phenylpyruvate decarboxylase in complex with its substrates and a covalent reaction intermediate analogue. These structures reveal the regulatory site and unveil the mechanism of allosteric substrate activation. This signal transduction relies on quaternary structure reorganizations, domain rotations, and a pathway of local conformational changes that are relayed from the regulatory site to the active site. The current findings thus uncover the molecular mechanism by which the binding of a substrate in the regulatory site is linked to the mounting of the catalytic machinery in the active site in this thiamine diphosphate-dependent enzyme. Thiamine diphosphate-dependent enzymes are involved in a wide variety of metabolic pathways. The molecular mechanism behind active site communication and substrate activation, observed in some of these enzymes, has since long been an area of debate. Here, we report the crystal structures of a phenylpyruvate decarboxylase in complex with its substrates and a covalent reaction intermediate analogue. These structures reveal the regulatory site and unveil the mechanism of allosteric substrate activation. This signal transduction relies on quaternary structure reorganizations, domain rotations, and a pathway of local conformational changes that are relayed from the regulatory site to the active site. The current findings thus uncover the molecular mechanism by which the binding of a substrate in the regulatory site is linked to the mounting of the catalytic machinery in the active site in this thiamine diphosphate-dependent enzyme. Thiamine diphosphate (ThDP), 4The abbreviations used are: ThDP, thiamine diphosphate; PPDC, phenylpyruvate decarboxylase; PDC, pyruvate decarboxylase; IPDC, indolepyruvate decarboxylase; 3dThDP, 3-deaza-ThDP; 2HE3dThDP, 2-(1-hydroxyethyl)-3-deaza-ThDP; PPA, phenylpyruvic acid; POVA, 5-phenyl-2-oxovaleric acid; POX, pyruvate oxidase. 4The abbreviations used are: ThDP, thiamine diphosphate; PPDC, phenylpyruvate decarboxylase; PDC, pyruvate decarboxylase; IPDC, indolepyruvate decarboxylase; 3dThDP, 3-deaza-ThDP; 2HE3dThDP, 2-(1-hydroxyethyl)-3-deaza-ThDP; PPA, phenylpyruvic acid; POVA, 5-phenyl-2-oxovaleric acid; POX, pyruvate oxidase. the biologically active form of vitamin B1, is an essential cofactor for a wide variety of enzymes that mainly mediate the making and breaking of carbon-carbon bonds adjacent to a carbonyl group (Fig. 1) (1Schowen R.L. Sinnott M. Comprehensive Biological Catalysis, A Mechanistic Reference. Vol. II. Academic Press, San Diego1998: 217-266Google Scholar). Although ThDP-dependent enzymes are textbook examples of cofactor-aided catalysis, the fine details of their catalytic mechanism are still lacking (2Jordan F. Nat. Prod. Rep. 2003; 20: 184-201Crossref PubMed Scopus (197) Google Scholar). Moreover, two types of intramolecular signaling have been observed in ThDP-dependent enzymes. Whereas substrate activation results from communication between an allosteric regulatory site and the active site, the communication among active sites leads to alternating site reactivity (3Sergienko E.A. Jordan F. Biochemistry. 2002; 41: 3952-3967Crossref PubMed Scopus (33) Google Scholar, 4Jordan F. Nemeria N. Sergienko E.A. Acc. Chem. Res. 2005; 38: 755-763Crossref PubMed Scopus (28) Google Scholar, 5Frank R.A. Titman C.M. Pratap J.V. Luisi B.F. Perham R.N. Science. 2004; 306: 872-876Crossref PubMed Scopus (145) Google Scholar). The molecular mechanism of substrate activation in ThDP-dependent enzymes has been an area of intensive research and debate ever since its first observation in 1967 (6Davies D.D. Biochem. J. 1967; 104: 50PCrossref PubMed Scopus (4) Google Scholar).Phenylpyruvate decarboxylase (PPDC) catalyzes the ThDP-mediated non-oxidative decarboxylation of phenyl- and indolepyruvate to phenyl- or indoleacetaldehyde and carbon dioxide (7Spaepen S. Versées W. Gocke D. Pohl M. Steyaert J. Vanderleyden J. J. Bacteriol. 2007; 189: 7626-7633Crossref PubMed Scopus (80) Google Scholar). In the root-associated bacterium Azospirillum brasilense, AbPPDC catalyzes the second step in the conversion of phenylalanine and tryptophan into phenyl- and indole-acetic acid, respectively (8Somers E. Ptacek D. Gysegom P. Srinivasan M. Vanderleyden J. Appl. Environ. Microb. 2005; 71: 1803-1810Crossref PubMed Scopus (99) Google Scholar, 9Costacurta A. Keijers V. Vanderleyden J. Mol. Gen. Genet. 1994; 243: 463-472Crossref PubMed Scopus (132) Google Scholar). The latter compound is a plant hormone, which is responsible for the plant growth promoting abilities of A. brasilense (10Dobbelaere S. Croonenborghs A. Thys A. Vande Broek A. Vanderleyden J. Plant Soil. 1999; 212: 155-164Crossref Google Scholar). AbPPDC shows high structural similarity to other 2-ketoacid decarboxylases from the pyruvate oxidase family (11Duggleby R.G. Acc. Chem. Res. 2006; 39: 550-557Crossref PubMed Scopus (74) Google Scholar), such as pyruvate decarboxylase (PDC) and indolepyruvate decarboxylase (IPDC) (12Versées W. Spaepen S. Vanderleyden J. Steyaert J. FEBS J. 2007; 274: 2363-2375Crossref PubMed Scopus (31) Google Scholar). Similar to several PDCs, the AbPPDC displays substrate activation with indolepyruvate and other substrates, characterized by sigmoidal v versus [S] plots (7Spaepen S. Versées W. Gocke D. Pohl M. Steyaert J. Vanderleyden J. J. Bacteriol. 2007; 189: 7626-7633Crossref PubMed Scopus (80) Google Scholar).Here we present a series of crystal structures of the ThDP-dependent phenylpyruvate decarboxylase in complex with different substrates and an analogue of a covalent reaction intermediate. These provide new snapshots along the reaction coordinate and unveil the regulatory site and a detailed molecular mechanism for allosteric substrate activation.EXPERIMENTAL PROCEDURESProtein Expression, Purification, and Preparation of the Complexes—The wild-type AbPPDC was cloned, expressed, and purified as described previously (7Spaepen S. Versées W. Gocke D. Pohl M. Steyaert J. Vanderleyden J. J. Bacteriol. 2007; 189: 7626-7633Crossref PubMed Scopus (80) Google Scholar, 12Versées W. Spaepen S. Vanderleyden J. Steyaert J. FEBS J. 2007; 274: 2363-2375Crossref PubMed Scopus (31) Google Scholar). During purification the enzyme activity was monitored with phenylpyruvate as substrate using the established coupled optical test with horse liver alcohol dehydrogenase and NADH (13Weiss P.M. Garcia G.A. Kenyon G.L. Cleland W.W. Cook P.F. Biochemistry. 1988; 27: 2197-2205Crossref PubMed Scopus (60) Google Scholar).Complexes of PPDC with the inhibitors 3-deaza-ThDP (PPDC-3dThDP) and 2-(1-hydroxyethyl)-3-deaza-ThDP (PPDC-2HE3dThDP) were subsequently obtained by incubating PPDC (purified without addition of ThDP) with 1 mm 3dThDP or 2HE3dThDP for 48 h at 4 °C. Complete exchange of ThDP with the analogues was confirmed by total loss of activity with phenylpyruvate as a substrate. The tertiary complexes with phenylpyruvic acid (PPDC-3dThDP-PPA) and 5-phenyl-2-oxovaleric acid (PPDC-3dThDP-POVA) were then obtained by incubating the PPDC-3dThDP complex with 100 mm phenylpyruvate and 5 mm 5-phenyl-2-oxovaleric acid, respectively.Crystallization and Data Collection—The PPDC-2HE3dThDP complex was crystallized by the hanging drop vapor diffusion method. Equal volumes of protein solution and precipitant containing 15% polyethylene glycol 4000 (w/v), 10% glycerol (v/v) in 100 mm Hepes buffer, pH 7.0, were mixed and equilibrated at 293 K. The crystals were transferred to a cryo-solution containing 20% polyethylene glycol 4000 (w/v), 25% glycerol (v/v) in 100 mm Hepes, pH 7.0, and transferred immediately to the cryostream. X-ray diffraction data were collected at 100 K to a resolution of 1.85 Å on beamline X11 (EMBL, DESY, Hamburg) using an x-ray wavelength of 0.8157 Å.The PPDC-3dThDP, PPDC-3dThDP-PPA, and PPDC-3dThDP-POVA complexes were crystallized at 293 K using the hanging drop vapor diffusion method with 11% (w/v) polyethylene glycol 3350 and 0.2 m di-ammonium tartrate, pH 6.5, as precipitant solution. Crystals were transferred to a cryo-solution containing 16% (w/v) polyethylene glycol 3350, 0.2 m diammonium tartrate, pH 6.5, 30% glycerol (with addition of PPA and POVA in case of the ternary complexes), and transferred immediately to the cryostream. X-ray diffraction data were collected at 100 K to a resolution of 3.2 Å on beamline X11 (EMBL, DESY, Hamburg) using an x-ray wavelength of 0.8162 Å for PPDC-3dThDP, to a resolution of 2.15 Å on beamline X11 (EMBL, DESY, Hamburg) using an x-ray wavelength of 0.8162 Å for PPDC-3DThDP-PPA; and to a resolution of 1.9 Å on beamline BW7A (EMBL, DESY, Hamburg) using an x-ray wavelength of 0.9732 Å for PPDC-3dThDP-POVA.The diffraction data were indexed and integrated using DENZO and scaled using SCALEPACK (14Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38361) Google Scholar). Intensities were converted to structure factor amplitudes using TRUNCATE (15French S. Wilson K. Acta Crystallogr. Sect. A. 1978; 34: 517-525Crossref Scopus (888) Google Scholar). Table 1 summarizes the data collection and processing statistics.TABLE 1Data collection and refinement statisticsPPDC-3dThDPPPDC-2HE3dThDPPPDC-3dThDP-PPAPPDC-3dThDP-POVAData collectionSpace groupC2221C2221C2221C2221Cell dimensionsa, b, c (Å)100.54, 179.81, 121.0399.98, 179.05, 120.8674.90, 145.58, 194.0474.69, 145.12, 194.11a, b, γ (°)90, 90, 9090, 90, 9090, 90, 9090, 90, 90Resolution (Å)50.0-3.2 (3.31-3.20)aHighest resolution shell is shown in parentheses.50.0-1.85 (1.92-1.85)50.0-2.15 (2.23-2.15)50.0-1.90 (1.97-1.90)Rsym0.16 (0.65)0.084 (0.44)0.11 (0.38)0.074 (0.48)I/σI11.0 (3.0)24.1 (3.9)20.8 (4.7)18.4 (2.45)Completeness (%)97.5 (98.3)98.3 (96.0)92.8 (83.6)92.5 (92.0)Redundancy5.9 (5.9)7.5 (6.8)8.4 (8.1)4.8 (2.5)RefinementResolution (Å)50.0-3.250.0-1.8550.0-2.1550.0-1.90No. reflections17,86590,95454,18276,981Rwork/Rfree17.61/28.8917.04/20.4318.21/23.9916.32/20.63No. atomsProtein7,8197,9437,9157,975Ligand/ion54132108132Water1351,0107391,117B-factorsProtein41.423.039.125.1Ligand/ion28.816.444.026.0Water24.235.148.440.2Root mean square deviationsBond lengths (Å)0.00700.00890.00900.0090Bond angles (°)1.3131.4581.4071.430Ramachandran plot (% in favored, allowed regions)88.4, 98.397.6, 99.597.9, 99.898.7, 99.7a Highest resolution shell is shown in parentheses. Open table in a new tab Structure Determination and Refinement—Initial phases for all complexes were obtained by molecular replacement with the program PHASER (16McCoy A.J. Grosse-Kunstleve R.W. Storoni L.C. Read R.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 2005; 61: 458-464Crossref PubMed Scopus (1596) Google Scholar) using a monomer of PPDC-ThDP (Protein Data Bank 2NXW) as a search model. The solutions were subjected to the simulated annealing procedure as implemented in CNS, and manual model building and inspection of electron density was performed in COOT (17Emsley P. Cowtan K. Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (22799) Google Scholar). After several cycles of positional and temperature factor refinement using CNS combined with manual corrections, solvent molecules, cofactors, and alternative conformations were included in the models. Structure refinement was considered complete after crystallographic R-factor and free R-factor had converged, and the difference density was without interpretable features. The final models were checked with the Molprobity web server (18Lovell S.C. Davis I.W. Adrendall 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 (3790) Google Scholar). Refinement statistics are summarized in Table 1. The structural superpositions were performed using the DALI server (19Holm L. Sander C. Nucleic Acids Res. 1998; 26: 316-319Crossref PubMed Scopus (595) Google Scholar) and/or the program LSQMAN (20Collaborative Computational Project Number 4Acta Crystallogr. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19704) Google Scholar). Figures were prepared with PyMOL (21DeLano W.L. The PyMOL Molecular Graphics System. DeLano Scientific, San Carlos, CA2002Google Scholar) and Molscript (22Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar).RESULTSSubstrate and Covalent Intermediate Complexes of PPDC—A standard procedure to obtain detailed structural information on the catalytic mechanism of an enzyme is to solve the structure of slow mutants with trapped substrates or covalent reaction intermediates. In a different approach we used unreactive analogues of the coenzyme to trap intermediates in a thiamine-dependent enzyme. 3dThDP is such an unreactive analogue of ThDP, in which the single nitrogen atom of the thiazolium ring is replaced by a carbon (23Mann S. Perez Melero C. Hawksley D. Leeper F.J. Org. Biomol. Chem. 2004; 2: 1732-1741Crossref PubMed Scopus (31) Google Scholar). Although an excellent steric mimic of the cofactor, 3dThDP electrostatically more closely resembles the overall neutral ylide form of thiamine, due to the absence of the positive charge on position 3. AbPPDC in which the naturally occurring ThDP is replaced by 3dThDP proves indeed to be enzymatically inert.The crystal structure of PPDC in complex with 3dThDP (PPDC-3dTHDP) was solved to 3.2-Å resolution. This structure is nearly perfectly superimposable on the structure of the native PPDC holo-enzyme (PPDC-ThDP) that we previously solved, showing that replacement of ThDP with 3dThDP causes no structural rearrangements (12Versées W. Spaepen S. Vanderleyden J. Steyaert J. FEBS J. 2007; 274: 2363-2375Crossref PubMed Scopus (31) Google Scholar) (see supplemental materials for all structural details). Because PPDC-3dThDP is enzymatically inert we were able to trap the substrates PPA and POVA in its active site and to solve the structures of these ternary complexes to 2.15-Å (PPDC-3dThDP-PPA) and 1.9-Å (PPDC-3dThDP-POVA) resolution, respectively. Chemical synthesis also allows 3dThDP to be substituted on the C2 to obtain stable analogues of covalent reaction intermediates in the reaction cycle (24Leeper F.J. Hawksley D. Mann S. Perez Melero C. Wood M. D.H. Biochem. Soc. Trans. 2005; 33: 772-775Crossref PubMed Scopus (14) Google Scholar). Using such an analogue we solved the structure of PPDC in complex with 2-(1-hydroxyethyl)-3-deaza-ThDP (PPDC-2HE3dThDP) to 1.85-Å resolution to study the structure of the last covalent intermediate on the reaction coordinate (Fig. 1, intermediate 5).All structures show clear electron density with full occupancy for all ligands bound to both active sites of the homodimer in the asymmetric unit (Fig. 2). In the PPDC-2HE3dThDP structure (Fig. 2b) the density for the 1-hydroxyethyl moiety of the intermediate analogue corresponds to a mixture of the R and S enantiomer at the Cα atom, consistent with the fact that a racemic mixture was used in the co-crystallization. Both enantiomers were modeled with half-occupancy. The overall structure of the active site is very similar in PPDC-3dThDP and PPDC-2HE3dThDP compared with the native holo-enzyme (Fig. 2, a and b). These three structures all exhibit an open active site with the active site loop spanning residues 104-120, completely disordered (see Versées et al. (12Versées W. Spaepen S. Vanderleyden J. Steyaert J. FEBS J. 2007; 274: 2363-2375Crossref PubMed Scopus (31) Google Scholar) for a detailed description of the PPDC-ThDP active site). In PPDC-3dThDP-PPA and PPDC-3dThDP-POVA, binding of the substrates is accompanied by a complete structuring of the 104-120 loop. Upon substrate binding, this long loop folds over the active site bringing His112 and His113 into the active site pocket (Fig. 2, c and d). Concomitantly, a second active site loop spanning residues 280-294 of the neighboring subunit of the tight dimer is reorganized, wedging residues Asp282 and Thr283 deeper into the active site pocket. This structural transition causes a H-bond to be formed between His112 of the first loop and Asp282 of the second loop. The reorganization of the 280-294 loop also permits new interactions with the C-terminal helix (e.g. between the side chain of Gln536 and Arg538 and the main chain carbonyls of Asp282 and Ala287 and Ser288, respectively) allowing this helix to bend over the active site.FIGURE 2Close up view of the active sites of PPDC-3dThDP (a), PPDC-2HE3dThDP (b), PPDC-3dThDP-PPA (c), and PPDC-3dThDP-POVA (d). Residues provided by different subunits are color-coded green and cyan. Parts of the 104-120 and 280-294 loops, and the C-terminal helix are shown in a coil representation. Cofactor and intermediate analogues are colored yellow, and substrates magenta. The Mg2+ ion and a protein bound water molecule are represented by a gray and red sphere, respectively. Interactions with the substrate and the water molecule are indicated by orange dotted lines. The electron density (contoured at 5 σ for PPDC-2HE3dThDP and PPDC-3dThDP-POVA, and 4 σ for PPDC-3dThDP and PPDC-3dThDP-PPA) of a Fo - Fc simulated annealed omit map calculated without cofactors and substrates is shown (blue mesh).View Large Image Figure ViewerDownload Hi-res image Download (PPT)A Separate, Regulatory Substrate-binding Site—Apart from the substrate molecules bound in the active sites, the PPDC-3dThDP-PPA and PPDC-3dThDP-POVA structures unambiguously reveal a second site of bound substrate in each subunit. This site is located at a distance of 18 Å from the active site, at the interface of the PYR, R, and PP domains of each subunit. The substrate molecules in this second site are tightly bound by residues coming from the three domains (Fig. 3). In both structures, the carboxyl group of the substrate forms charged H-bonds with the Arg60 and Arg215 (bidentate) side chains and an additional H-bond to the main chain amide of Ala397. A third arginine residue, Arg214, is involved in a cation-π stacking interaction with the phenyl moiety of the substrate. The 2-keto oxygen of the substrate forms an H-bond with a protein-bound water molecule. Remarkably, this carbonyl oxygen makes very close (unfavorable) contacts with the main chain carbonyls of Met238 (2.9 Å), Arg240 (2.8 Å), and Leu395 (3.0 Å).FIGURE 3The regulatory substrate-binding site of PPDC-3dThDP-POVA: location of the regulatory substrate-binding site at the juncture of the three domains in a PPDC subunit (a), and close up view of the interactions with the regulatory substrate (b). The PYR, R, and PP domains in a are color-coded blue, green, and red, respectively, and the same coloring is maintained in b for the C-atoms of the amino acid residues in the regulatory site. The POVA molecules bound in the active site and in the regulatory site are shown as sticks and colored magenta and orange, respectively. In panel b the electron density (contoured at 5 σ) of a Fo - Fc simulated annealed omit map calculated around the regulatory POVA molecule is shown as a blue mesh.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Tertiary Structure Rearrangements upon Substrate Binding—The asymmetric units of all PPDC structures contain a homodimer. In this dimer, each subunit adopts the archetypical pyruvate oxidase (POX) fold consisting of the PYR, R, and PP domains (25Muller Y.A. Schulz G.E. Science. 1993; 259: 965-967Crossref PubMed Scopus (215) Google Scholar). The secondary complexes PPDC-ThDP, PPDC-3dThDP, and PPDC-2HE3dThDP are very similar in subunit architecture (see supplemental Table 1S for a full list of root mean square deviations upon superposition). Upon substrate binding in the active and regulatory sites, a change in this subunit architecture involving a domain rotation is observed in both ternary complexes (PPDC-3dThDP-PPA and PPDC-3dThDP-POVA). When the PYR and PP domains of PPDC-ThDP and either of the substrate complexes are superimposed, the R domains differ by a rotation of about 12° (Fig. 4). This domain rotation is accompanied by a large rearrangement of active site loop 280-294 of the R domain and the concomitant ordering of active site loop 104-120 of the PYR domain of the neighboring subunit causing the complete closure of the active sites in PPDC-3dThDP-PPA and PPDC-3dThDP-POVA.FIGURE 4Tertiary structure rearrangements upon substrate binding. A superposition of the binary complex PPDC-ThDP and the ternary complex PPDC-3dThDP-POVA is shown, zoomed in on one subunit. Whereas the PYR and PP domains of PPDC-ThDP and PPDC-3dThDP-POVA superimpose nearly perfectly, the R domain rotates by about 12° upon substrate binding. PPDC-ThDP is shown in gray; PPDC-3dThDP-POVA is colored according to the domain, with the PYR, R, PP domains, and the intervening loops colored blue, green, red, and yellow, respectively. The mobile 280-294 loop of the R domain and the 104-120 loop of the PYR domain of the neighboring subunit are colored orange and are indicated by arrows. His112 and His113 of loop 104-120 and Asp282 of loop 280-294 are represented as sticks.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Quaternary Structure Rearrangements upon Substrate Binding—PPDC adopts a homotetrameric assembly in solution (7Spaepen S. Versées W. Gocke D. Pohl M. Steyaert J. Vanderleyden J. J. Bacteriol. 2007; 189: 7626-7633Crossref PubMed Scopus (80) Google Scholar). In all the crystal structures, the two tight dimers constituting the biological tetramers are related through a 2-fold crystallographic symmetry axis (7Spaepen S. Versées W. Gocke D. Pohl M. Steyaert J. Vanderleyden J. J. Bacteriol. 2007; 189: 7626-7633Crossref PubMed Scopus (80) Google Scholar, 12Versées W. Spaepen S. Vanderleyden J. Steyaert J. FEBS J. 2007; 274: 2363-2375Crossref PubMed Scopus (31) Google Scholar). However, large differences exist between the tetramer architecture of the binary complexes (PPDC-ThDP, PPDC-3dThDP, and PPDC-2HE3dThDP) and the ternary complexes with substrates bound at the active and the regulatory sites (PPDC-3dThDP-PPA and PPDC-3dThDP-POVA). In the binary complexes, the non-perpendicular arrangement of non-crystallographic axes relating the monomers in the asymmetric unit and the crystallographic axis relating the two dimers results in an asymmetrical tetramer assembly, best described as an asymmetrical dimer of dimers (Fig. 5a). For the ternary complexes the non-crystallographic symmetry axes relating the two subunits in the dimer intersect with the crystallographic axis at an angle of 90°, resulting in a dimer of dimers with pseudo 222 symmetry (Fig. 5b). In going from the asymmetrical dimer of dimers observed for the binary complexes to the symmetrical dimer of dimers of the ternary complexes, one dimer has to be rotated by about 34°, vis à vis to the second dimer. This also has implications for the dimer-dimer interfaces. Whereas the AC and BD interfaces are different in the asymmetrical tetramers (see Fig. 5, for subunit nomenclature) these interfaces are the same for the symmetrical tetramers.FIGURE 5Quaternary structure rearrangements upon substrate binding. The biological tetramers of the binary complex PPDC-2HE3dThDP (a) and the ternary complex PPDC-3dThDP-POVA (b) are depicted with their AB homodimer in the same orientation. The transition from an asymmetrical dimer of dimers in substrate-free PPDC to a symmetrical dimer of dimers in substrate-bound PPDC is accompanied by a 34° rotation of the CD dimer vis à vis the AB dimer. Cofactors and substrates are shown as sticks with the carbon atoms of 3dThDP and 2HE3dTHDP colored yellow and the POVA substrates colored purple. The crystallographic 2-fold axis relating the two dimers is indicated in orange. The two NCS-axes relating the subunits in the asymmetric unit are indicated as blue and red bars.View Large Image Figure ViewerDownload Hi-res image Download (PPT)A Cascade of Conformational Changes Links the Regulatory Substrate-binding Site to the Active Site—Comparison of the binary complexes (PPDC-ThDP, PPDC-3dThDP, and PPDC-2HE3dThDP) with the ternary complexes (PPDC-3dThDP-PPA and PPDC-3dThDP-POVA) uncovers a series of coupled local conformational changes running from the regulatory substrate-binding site to the active site (see Fig. 6). Binding of a substrate molecule in the regulatory site causes a change in side chain conformation of Arg214 leading to the formation of a cation-π interaction with the phenyl group of the substrate. The void left by the arginine side chain gets occupied by the side chain of Leu242. This rear-rangement of Leu242, together with the steric repulsion caused by the binding of the 2-ketoacid moiety of the substrate and the loss of the H-bond between Arg214 and the main chain carbonyl of Arg240, induces a relocation of the entire region between Phe237 and Pro247. The changes in the Phe237-Pro247 segment lead to further structural rearrangements in two distinct branches both leading to the active site. In a first branch, changes at Met238 force the Tyr400 side chain to flip around with a concomitant displacement of its hydroxyl group by 9.8 Å. This conformational change in its turn forces a relocation of Leu109 of the neighboring subunit and the associated structuring of the entire 104-120 loop, bringing the two residues His112 and His113 into the active site. The second branch of the cascade is initiated by the steric clash of Phe237 with Phe285 located on the 280-294 active site loop. This causes a rearrangement of this entire loop, the most obvious change being a 13-Å displacement of the Phe285 side chain. Reorganization of the 280-294 loop (branch 2) liberates the necessary space for the 104-120 loop (branch 1) to enter the active site. Asp282 is also shifted into the active site in this process (a shift of 5 Å) and now forms a H-bond with His112. This cascade of transmitted conformational changes clearly provides a link between substrate binding in the regulatory substrate-binding site and structural changes in the active site.FIGURE 6Intramolecular signal transduction via a cascade of conformational changes. A superposition of the relevant residues of the unactivated binary complex PPDC-ThDP (in gray) with the activated ternary complex PPDC-3dThDP-POVA (in green and blue for residues of the A and B subunit, respectively) is shown. POVA molecules in the regulatory and active site are shown in magenta. Binding of a substrate molecule in the regulatory site causes a rearrangement of Arg214, with concomitant rearrangement of the region between Phe237 and Leu242. These rearrangements are transmitted via Tyr400 and Phe285 to the 104-120 and 280-294 loops that fold over the active site. A H-bond between Asp282 and His112 is hereby formed completing the proposed Asp25-His112-Asp282 catalytic triad. This sterical relay mechanism hence provides a clear link between substrate binding in the regulatory site and catalytic competence in the active site.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONSnapshots along the Catalytic Cycle of a ThDP-dependent Decarboxylase—Multiple studies have been reported dealing with the complex kinetics of ThDP-dependent enzymes. However, a full interpretation of this wealth of data has always been hampered by a lack of structures visualizing the relevant reaction intermediates. Only recently structures of some of these intermediates have been solved: the lactyl-ThDP intermediate in the active site of pyruvate oxidase (intermediate 3, see Fig. 1); the planar enamine intermediate in the active site of pyruvate oxidase and transketolase (intermediate 4B, see Fig. 1) and the non-planar, more carbanion-like form of the latter intermediate in the active site of branched-chain 2-ketoacid dehydrogenase (intermediate 4A, see Fig. 1) (26Wille G. Meyer D. Steinmetz A. Hinze E. Golbik R. Tittmann K. Nat. Chem. Biol. 2006; 2: 324-328Crossref PubMed Scopus (98) Google Scholar, 27Fiedler E. Thorell S. Sandalova T. Golbik R. Konig S. Schneider G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 591-595Crossref PubMed Scopus (107) Google Scholar, 28Machius M. Wynn R.M. Chuang J.L. Li J. Kluger R. Yu D. Tomchick D.R. Brautigam C.A. Chuang D.T. Structure. 2006; 14: 287-298Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). In the current paper we present the crystal structures of two other reaction intermediates. The use of the non-reactive cofactor analogue 3-deaza-ThDP allowed to solve structures in complex with the genuine substrates phenylpyruvic acid and 5-phenyl-2-oxovaleric acid to high resolution, providing images of the enzyme-substrate Michaelis complex for this class of enzymes (intermediate 2, see Fig. 1). Moreover, the structure of PPDC in complex with 2-(1-hydroxyethyl)-3-deaza-ThDP p" @default.
• W2003013017 created "2016-06-24" @default.
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• W2003013017 date "2007-11-01" @default.
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• W2003013017 title "Molecular Mechanism of Allosteric Substrate Activation in a Thiamine Diphosphate-dependent Decarboxylase" @default.
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