Title: A Thiamin-bound, Pre-decarboxylation Reaction Intermediate Analogue in the Pyruvate Dehydrogenase E1 Subunit Induces Large Scale Disorder-to-Order Transformations in the Enzyme and Reveals Novel Structural Features in the Covalently Bound Adduct
Abstract: The crystal structure of the E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc) has been determined with phosphonolactylthiamin diphosphate (PLThDP) in its active site. PLThDP serves as a structural and electrostatic analogue of the natural intermediate α-lactylthiamin diphosphate (LThDP), in which the carboxylate from the natural substrate pyruvate is replaced by a phosphonate group. This represents the first example of an experimentally determined, three-dimensional structure of a thiamin diphosphate (ThDP)-dependent enzyme containing a covalently bound, pre-decarboxylation reaction intermediate analogue and should serve as a model for the corresponding intermediates in other ThDP-dependent decarboxylases. Regarding the PDHc-specific reaction, the presence of PLThDP induces large scale conformational changes in the enzyme. In conjunction with the E1-PLThDP and E1-ThDP structures, analysis of a H407A E1-PLThDP variant structure shows that an interaction between His-407 and PLThDP is essential for stabilization of two loop regions in the active site that are otherwise disordered in the absence of intermediate analogue. This ordering completes formation of the active site and creates a new ordered surface likely involved in interactions with the lipoyl domains of E2s within the PDHc complex. The tetrahedral intermediate analogue is tightly held in the active site through direct hydrogen bonds to residues His-407, Tyr-599, and His-640 and reveals a new, enzyme-induced, strain-related feature that appears to aid in the decarboxylation process. This feature is almost certainly present in all ThDP-dependent decarboxylases; thus its inclusion in our understanding of general thiamin catalysis is important. The crystal structure of the E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc) has been determined with phosphonolactylthiamin diphosphate (PLThDP) in its active site. PLThDP serves as a structural and electrostatic analogue of the natural intermediate α-lactylthiamin diphosphate (LThDP), in which the carboxylate from the natural substrate pyruvate is replaced by a phosphonate group. This represents the first example of an experimentally determined, three-dimensional structure of a thiamin diphosphate (ThDP)-dependent enzyme containing a covalently bound, pre-decarboxylation reaction intermediate analogue and should serve as a model for the corresponding intermediates in other ThDP-dependent decarboxylases. Regarding the PDHc-specific reaction, the presence of PLThDP induces large scale conformational changes in the enzyme. In conjunction with the E1-PLThDP and E1-ThDP structures, analysis of a H407A E1-PLThDP variant structure shows that an interaction between His-407 and PLThDP is essential for stabilization of two loop regions in the active site that are otherwise disordered in the absence of intermediate analogue. This ordering completes formation of the active site and creates a new ordered surface likely involved in interactions with the lipoyl domains of E2s within the PDHc complex. The tetrahedral intermediate analogue is tightly held in the active site through direct hydrogen bonds to residues His-407, Tyr-599, and His-640 and reveals a new, enzyme-induced, strain-related feature that appears to aid in the decarboxylation process. This feature is almost certainly present in all ThDP-dependent decarboxylases; thus its inclusion in our understanding of general thiamin catalysis is important. Thiamin-dependent enzymes play key roles in sugar metabolism, typically catalyzing the decarboxylation of α-keto acids and the transfer of an aldehyde or an acyl group (1Kluger R. Chem. Rev. 1987; 87: 863-876Crossref Scopus (250) Google Scholar, 2Reed L. Acc. Chem. Res. 1974; 7: 40-46Crossref Scopus (654) Google Scholar, 3Jordan F. Nat. Prod. Rep. 2003; 20: 184-201Crossref PubMed Scopus (201) Google Scholar, 4Furey W. Arjunan P. Brunskill A. Chandrasekhar K. Nemeria N. Wei W. Yan Y. Zhang A. Jordan F. Jordan F. Patel M.S. Thiamine: Catalytic Mechanisms and Role in Normal and Disease States. Marcel Dekker, Inc., New York2004: 407-432Google Scholar, 5Jordan F. Nemeria N. Bioorg. Chem. 2005; 33: 190-215Crossref PubMed Scopus (45) Google Scholar). Examples include the E1 components in pyruvate dehydrogenase complexes (PDHc), 2The abbreviations used are: PDHc, pyruvate dehydrogenase multienzyme complex; ThDP, thiamin diphosphate (vitamin B1 diphosphate); LThDP, lactylthiamin diphosphate (reaction intermediate); PLThDP, phosphonolactylthiamin diphosphate (reaction intermediate analogue); E1, first enzymatic component of multienzyme complexes related to and including PDHc; parental E1, protein only component of E1; E1-ThDP, parental E1 with ThDP cofactor; E1-PLThDP, parental E1 with PLThDP; H407A E1-PLThDP, H407A parental E1 variant with PLThDP. pyruvate decarboxylase, transketolase, etc. Crystallographic studies (6Arjunan P. Umland T. Dyda F. Swaminathan S. Furey W. Sax M. Farrenkopf B. Gao Y. Zang D. Jordan F. J. Mol. Biol. 1996; 256: 590-600Crossref PubMed Scopus (192) Google Scholar, 7Dyda F. Furey W. Swaminathan S. Sax M. Farrenkopf B. Jordan F. Biochemistry. 1993; 32: 6165-6170Crossref PubMed Scopus (226) Google Scholar, 8Arjunan P. Nemeria N. Brunskill A. Chandrasekhar K. Sax M. Yan Y. Jordan F. Guest J.R. Furey W. Biochemistry. 2002; 41: 5213-5221Crossref PubMed Scopus (123) Google Scholar, 9Aevarsson A. Seger K. Turley S. Sokatch J. Hol W. Nat. Struct. Biol. 1999; 6: 785-792Crossref PubMed Scopus (121) Google Scholar, 10Muller Y.A. Lindqvist Y. Furey W. Schulz G.E. Jordan F. Schneider G. Structure (Camb.). 1993; 1: 95-103Abstract Full Text PDF PubMed Scopus (174) Google Scholar, 11Fiedler E. Thorell S. Sandalova T. Golbik R. Konig S. Schneider G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 591-595Crossref PubMed Scopus (109) Google Scholar, 12Chabriere E. Charon M. Volbeda A. Pieulle L. Hatchikian E. Fontecilla-Camps J. Nat. Struct. Biol. 1999; 6: 182-190Crossref PubMed Scopus (165) Google Scholar) have elucidated many of the structural, stereochemical, and biochemical details in the mechanism of action of these enzymes and in the catalytic role of the cofactor ThDP (thiamin diphosphate, vitamin B1 diphosphate, Fig. 1, top left). Despite the enormous contributions made by these and other studies to our understanding of how such enzymes function, important details still remain obscure. There are, for example, no detailed structural data on the first ThDP-bound intermediate in the presence of any enzyme (for example, α-lactylthiamin diphosphate (α-LThDP) in PDHc E1 and pyruvate decarboxylase), which is postulated to form in the currently accepted mechanism of thiamin catalysis (Fig. 1, top, third object from the right). In an effort to obtain structural information pertaining to this key intermediate, we have determined the crystal structure of PDHc E1 from Escherichia coli in complex with α-phosphonolactylthiamin diphosphate (PLThDP). PLThDP is the product of the reaction between ThDP and methylacetylphosphonate, with the latter being an analogue of the true substrate pyruvate and a potent inhibitor of PDHc. The complex formed with PLThDP instead of ThDP therefore mimics the structure of the enzyme-bound, reactive tetrahedral intermediate α-LThDP (13Zhang S. Liu M. Yan Y. Zhang Z. Jordan F. J. Biol. Chem. 2004; 279: 54312-54318Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) in the decarboxylation step of the PDHc E1 reaction. It differs from the complex formed with the true substrate only in the replacement of the carboxylate group by a methyl phosphonate (PO3Me) group. However, unlike the C2α-CO2 bond normally cleaved in the reaction with pyruvate, the C2α-PO3Me bond remains intact. The reaction is therefore trapped in a pre-CO2 release-like state, and the structure represents a covalently bound, pre-decarboxylation reaction intermediate analogue. There have been three covalently bound reaction intermediate structures reported for ThDP-dependent enzymes (11Fiedler E. Thorell S. Sandalova T. Golbik R. Konig S. Schneider G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 591-595Crossref PubMed Scopus (109) Google Scholar, 12Chabriere E. Charon M. Volbeda A. Pieulle L. Hatchikian E. Fontecilla-Camps J. Nat. Struct. Biol. 1999; 6: 182-190Crossref PubMed Scopus (165) Google Scholar, 14Nakai T. Nakagawa N. Maoka N. Masui R. Kuramitsu S. Kamiya N. J. Mol. Biol. 2004; 337: 1011-1033Crossref PubMed Scopus (42) Google Scholar), but they all represented the planar enamine intermediate (Fig. 1, top right object) that exists only after decarboxylation. The E1-PLThDP structure is thus the first structural example of a covalently bound, pre-decarboxylation reaction intermediate analogue in any ThDP-dependent enzyme. The E1 component of the PDHc complex catalyzes the rate-limiting step of the overall PDHc reaction and therefore also provides an ideal target for mechanistic structural investigation. Initial crystallographic results for the E1-PLThDP complex have been reported previously (15Arjunan P. Sax M. Brunskill A. Nemeria N. Jordan F. Furey W. Acta Crystallogr. Sect. A. 2005; 61: C202Crossref Google Scholar), and complete, detailed results at 2.1 Ä resolution are now presented with the analysis revealing several unique structural features that are absent in the native enzyme. 1) The enzyme-bound conformation of PLThDP is the V form, rather than the S form determined many years ago for the same compound in the absence of enzyme, showing how the enzyme can enforce this highly strained V conformation even in the intermediate analogue. 2) The structure identifies a role for residue His-407, explaining previous biochemical data, and shows the direct interaction (and implied function) of several additional active center residues with the covalently bound adduct. 3) Major reorganization of the active center takes place and includes the ordering of two key loops not seen in any of our other PDHc E1 structures before; thus the PLThDP dramatically diminishes their mobility. 4) A marked deviation from co-planarity of the C2–C2α bond with the thiazolium ring is clearly revealed and suggests likely mechanistic consequences. 5) The structure shows strong interaction of the C2α-OH with the N4′ atom of the 4′-aminopyrimidine of ThDP, as evidenced by a short contact distance. Elsewhere, we used PLThDP in solution to show that PLThDP exists with the 1′,4′-iminoThDP tautomeric form (16Jordan F. Nemeria N. Zhang S. Yan Y. Arjunan P. Furey W. J. Am. Chem. Soc. 2003; 127: 12732-12738Crossref Scopus (67) Google Scholar, 17Nemeria N. Baykal A. Joseph E. Zhang S. Yan Y. Furey W. Jordan F. Biochemistry. 2004; 43: 6565-6575Crossref PubMed Scopus (89) Google Scholar) of the 4′-aminopyrimidine ring, the first instance in which this putative intermediate could be stabilized. The observed short O–N4′ distance is fully consistent with this tautomerization. The structural reorganization and ordering resulting from the presence of PLThDP deep in the active site extends to the enzyme exterior, providing a new surface likely involved in interactions with other enzymatic components within the PDHc multienzyme complex. Crystallization and Data Collection—The PDHc E1 from E. coli was purified and assayed according to published procedures (18Nemeria N. Yan Y. Zhang Z. Brown A.M. Arjunan P. Furey W. Guest J.R. Jordan F. J. Biol. Chem. 2001; 276: 45969-45978Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). ThDP, when present, was removed by gel filtration using an Amersham Biosciences G-25 column. For crystallization, the protein was dialyzed against 20 mm HEPES buffer at pH 7.0, containing 5 mm dithiothreitol, 0.2% NaN3, 0.5 mm EDTA, and 1 μm leupeptin. The H407A E1 variant was purified and assayed as reported previously (19Nemeria N. Arjunan P. Brunskill A. Sheibani F. Wei W. Yan Y. Zhang S. Jordan F. Furey W. Biochemistry. 2002; 41: 15459-15467Crossref PubMed Scopus (31) Google Scholar). The parental and corresponding variant E1 proteins were independently co-crystallized with PLThDP and Mg2+ by the sitting drop vapor diffusion method under conditions similar to that used for our earlier E1-ThDP crystallographic studies (8Arjunan P. Nemeria N. Brunskill A. Chandrasekhar K. Sax M. Yan Y. Jordan F. Guest J.R. Furey W. Biochemistry. 2002; 41: 5213-5221Crossref PubMed Scopus (123) Google Scholar). The best crystals were obtained at a reservoir solution with 15–20% polyethylene glycol 2000 monomethyl ether, 10% propanol, 0.2% NaN3, and 60 mm Hepes buffer (pH 7.05) at 22 °C. Drops were 6–10 μl consisting of equal parts of reservoir and protein solution. Crystal growth took about 4–6 weeks with crystals typically having dimensions of 0.1 × 0.20 × 0.30 mm. X-ray diffraction data for the E1-PLThDP complex were collected from a single crystal flash-cooled at –180°C on a Bruker HiSTAR detector, using a Rigaku RU200 rotating anode generator as source. An Osmic mirror system was used along with a thin nickel foil to provide a highly focused, intense beam of CuKα radiation. The crystals are monoclinic with space group P21 and cell constants a = 81.6, b = 142.5, c = 82.1 Ä, and β = 102.4°, with two molecules (subunits) in an asymmetric unit. To 2.1 Ä resolution, 466,210 total observations were reduced to yield 99,699 unique reflections (94% complete) with an internal R factor (based on intensities) of 0.07. The data were processed with the XGEN package (20Howard A.J. Gilliland G.L. Finzel B.C. Poulos T.L. Ohlendorf D.H. Salemme F.R. J. Appl. Crystallogr. 1987; 20: 383-387Crossref Scopus (571) Google Scholar) and scaled with a local version of Weissman's Fourier scaling program FBSCALE (21Weissman L. Computational Crystallography. Clarendon Press, Oxford1982: 56-63Google Scholar). For the H407A E1-PLThDP complex, synchrotron data were collected at SERCAT (Advanced Photon Source) and processed with the HKL2000 package (22Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38572) Google Scholar). Structure Determination and Refinement—Since the previous E1-ThDP crystals are isomorphous with both E1-PLThDP and H407A E1-PLThDP crystals (cell constants differing by less than 0.8 Ä), atomic coordinates from the former structure were used as the starting model for both refinements. The initial model included 1602 amino acids and omitted the ThDP cofactors. Following rigid body refinement, the E1-PLThDP model was further refined by simulated annealing using the program XPLOR (23Brunger A.T. Krukowski A. Erickson J.W. Acta Crystallogr. Sect. A. 1990; 46: 585-593Crossref PubMed Scopus (600) Google Scholar) without imposing any non-crystallographic symmetry restraints. After the initial refinement, examination of electron density difference maps clearly showed strong density for the intermediate analogue, PLThDP, and also produced strong and interpretable electron density for two of the three previously unobserved regions, 401–413 and 541–557. After including the PLThDP and the two missing regions, the model was refined with simulated annealing, and subsequent cycles consisted of positional and B factor refinement. The refinement procedure included periodic examinations of simulated annealing omit and difference maps, as well as the introduction of water molecules. A final examination of difference electron density maps still did not produce interpretable electron density for the N-terminal residues 1–55, and they are excluded from the model. Inspection of electron density maps, model building, and structural comparisons were carried out by using the program O (24Jones T.A. Zou J-Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13011) Google Scholar). For all data to 2.1 Ä resolution, the R factor is 0.203 (0.213 in the last shell), and the Rfree(based on 5.3% of data) is 0.254 (0.291 in the last shell). The final structure has good stereochemistry with root mean square deviations from ideality of 0.006 Ä and 1.3° for bond lengths and angles, respectively. The final model contains two independent subunits, each with 831 amino acids out of a possible 886, as well as two PLThDP-Mg+2 cofactor pairs. This model also includes 564 water molecules, five phosphate ions, and two cisprolines (Pro-463) per asymmetric unit. The model was analyzed with PROCHECK (25Laskowski R.A. MacArthur M.W. Moss M.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar), and 89% of the residues are in the most favored region in the Ramachandran plot (26Ramachandran G.N. Sasisekharan V. Adv. Protein Chem. 1968; 23: 283-437Crossref PubMed Scopus (2764) Google Scholar). The Ramachandran plot shows no residues in disallowed regions in either subunit. The structure of the H407A E1-PLThDP complex was determined and refined in a similar manner. However, in this structure, although the PLThDP was clearly present in strong density, the regions 1–55, 401–413, and 541–557 were completely disordered. The new structure was refined using the program CNS (27Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Price L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16967) Google Scholar). The final model, which included 550 water molecules, resulted in an R factor of 21.6% and a Rfree of 24.1% to 1.85 Ä resolution. The slightly higher R factor for the H407A E1-PLThDP variant may be attributed to the fact that this structure contains 60 disordered residues absent from the model (the regions 401–413 and 541–557 in each molecule) that were ordered in the E1-PLThDP structure. Ramachandran statistics are similar to those for the E1-PLThDP structure. All data collection and refinement statistics are given in Table 1. Graphical representations of protein models were generated by using the program RIBBONS (28Carson M. J. Appl. Crystallogr. 1991; 24: 958-961Crossref Scopus (783) Google Scholar). Coordinates and structure factors for the E1-PLThDP and H407A E1-PLThDP structures have been deposited in the Protein Data Bank with the access codes 2G25 and 2G28, respectively.TABLE 1Crystallographic data and refinement statisticsE1-PLThDPH407A E1-PLThDPSpace groupP21P21Unit cell a (Ä)81.682.0 b (Ä)142.5143.2 c (Ä)82.182.5 β (°)102.4102.6Resolution2.1 Ä1.55 ÄCompleteness % (last shell)94, (66)95, (33)Total reflections466,210685,515Unique reflections99,699213,020Rmerge0.0650.076Refinement statistics Resolution range (Ä)39.2-2.1042.0-1.85 Number of reflections93,771150,327 R factor (last shell)0.203 (0.213)0.216 (0.267) Rfree (last shell)0.254 (0.291)0.240 (0.300) Number of residues16621602 Number of waters564550Average B factor (Ä2) Main chain atoms9.5021.04 Side chain atoms11.1521.17 Solvent atoms16.023.68Root mean square deviations Bond lengths (Ä)0.0060.009 Bond angles (°)1.3°1.5° Open table in a new tab The main chain folds in PDHc E1-ThDP (8Arjunan P. Nemeria N. Brunskill A. Chandrasekhar K. Sax M. Yan Y. Jordan F. Guest J.R. Furey W. Biochemistry. 2002; 41: 5213-5221Crossref PubMed Scopus (123) Google Scholar) and in its E1-PLThDP counterpart are very similar, with both structures containing two subunits in an asymmetric unit. The two independent subunits are almost identical stereochemically and are related by a 2-fold non-crystallographic symmetry axis. The complete dimer is shown in Fig. 2a. Each E1-PLThDP subunit consists of a single polypeptide chain folded into three domains: the N terminus (1–470), the middle (471–700), and the C terminus (701–886). Least-squares fitting of 801 common α-carbon atoms in the E1-ThDP and E1-PLThDP structures results in a root mean square deviation of 0.40 Ä. However, significant main-chain deviations (up to 2.7 Ä) occur in the middle domain helix containing residues 525–535, which are involved in close contacts with residues present in the active site channel. Except for a few side chains at the surface of the protein, the electron density over the entire molecule is generally very well defined, but as in the E1-ThDP structure, there is no interpretable electron density for the N-terminal residues 1–55. A surprising observation in the E1-PLThDP map is the presence of well defined electron density for loop residues 401–413 and 541–557 that was absent in the map of the E1-ThDP structure due to complete disorder. These two loops form part of the active site channel situated at the dimer interface. The 401–413 loop forms part of the active site, and residue His-407 in this loop forms a critical hydrogen bond to an oxygen atom in the substrate analogue phosphonyl group. This loop stabilizes the other newly ordered loop 541–557, assisted by interactions created from the large movement in the helical region 525–535. The ordering creates new interface interactions between residues Lys-403–Asn-404 from one subunit and residues Gln-548–Asp-549 from the other subunit. The two newly ordered regions thus interact with each other and propagate outward from the active site to the enzyme surface. The newly ordered regions are shown in terms of the overall structure in Fig. 2a and in greater clarity regarding the active site and enzyme surface in Fig. 2b. Electron density for the 401–413 loop and its interactions with PLThDP are shown in Fig. 3. The average B value for main and side chain atoms is 9.5 and 11.2 Ä2, respectively. These values are significantly lower than those observed in the E1-ThDP structure (16.0 and 18.6 Ä2, respectively) (8Arjunan P. Nemeria N. Brunskill A. Chandrasekhar K. Sax M. Yan Y. Jordan F. Guest J.R. Furey W. Biochemistry. 2002; 41: 5213-5221Crossref PubMed Scopus (123) Google Scholar), implying a more ordered assembly. Accordingly, the presence of the reaction intermediate analogue in the active site supports the formation of a tighter E1 dimer. The now ordered loop regions provide a new exterior surface that can conceivably interact with other subunits of the PDHc complex and also partially seal the active site entrance, leading to a more hydrophobic catalytic region. It is important to note that the E1-ThDP and E1-PLThDP complex crystals are isomorphous; thus the ordering cannot be attributed to new packing contacts in a different crystal form and arises only from the presence of the adduct now covalently bound to the cofactor. The H407A E1-PLThDP structure (not shown) reveals the active site to be intact and the structure generally unchanged when compared with the E1-ThDP (8Arjunan P. Nemeria N. Brunskill A. Chandrasekhar K. Sax M. Yan Y. Jordan F. Guest J.R. Furey W. Biochemistry. 2002; 41: 5213-5221Crossref PubMed Scopus (123) Google Scholar) and E1-PLThDP structures. As in E1-ThDP and unlike in the E1-PLThDP structure, however, a final difference electron density map did not contain interpretable electron density for loop regions 401–413 and 541–557; the N-terminal region 1–55 is unobserved in all of the structures. When combined with the other structures, analysis of the H407A E1-PLThDP structure therefore confirmed the need for the His-407-PLThDP interaction in inducing the disorder-to-order transformation in two loops as the loops are ordered only when both the intermediate and His-407 are present. Clearly, the ordering is induced by the presence of PLThDP via direct hydrogen-bonding to residue His-407 and results in providing previously missing structural elements to complete both the exterior surface and the interior surface leading to the active sites. For E1-PLThDP, a difference electron density map calculated with the protein-only model indicated strong positive density for the PLThDP (Fig. 4). Funnel-shaped channels leading to the active sites are about 24 Ä deep, with walls formed by residues from both the N-terminal domain and the middle domain. In the absence of enzyme, a prior PLThDP crystal structure analysis (29Turano A. Furey W. Pletcher J. Sax M. Pike D. Kluger R. J. Am. Chem. Soc. 1982; 104: 3089-3095Crossref Scopus (33) Google Scholar) revealed that the thiamin portion of the molecule assumes the S conformation (Φt = –99.5°, Φp = –173.6°), which is characteristic of other C2-substituted thiamins and places C2 and its bound adduct distant from N4′ of the aminopyrimidine ring. The enzyme-bound PLThDP, however, adopts the V conformation with torsion angles (30Pletcher J. Sax M. Blank G. Wood M. J. Am. Chem. Soc. 1977; 99: 1396-1403Crossref PubMed Scopus (90) Google Scholar) Φt and Φp of 100.5 and –67°, respectively, and results in crowding near C2. The C2α-PO3Me bond is directed very nearly perpendicular to the thiazolium ring. The PLThDP conformation is stabilized by hydrogen-bonding interactions with Tyr-599, His-640, His-407, and Gln-408 (through a water). The active site cleft is lined with residues His-106, Ser-109, Gln-140, His-142, Tyr-177, Met-194, Asp-230, Glu-235, Asn-260, Leu-264, Lys-392, His-407, and Gln-408 from one subunit and residues Asp-521, Thr-525, Ile-569, Glu-571, Tyr-599, Phe-602, Glu-636, and His-640 from the other subunit. A stereo view of the dimer interface environment around the catalytic center is shown in Fig. 5. The structure reveals residues on the enzyme that are responsible for stabilization of the intermediate and that are capable of participating in catalysis as well. On one side of the thiazolium ring, in the vicinity of the covalent adduct on C2, is a cluster of four histidine residues. Of these, His-142 is involved in binding with the diphosphate group of the cofactor, whereas His-407 and His-640 interact with oxygen atoms in the adduct. The fourth, His-106, is bound to a water molecule. As seen earlier, His-407 is hydrogen-bonded to one of the phosphonyl oxygens, and His-640 is hydrogen-bonded to the C2α-hydroxyl oxygen atom. Tyr-599 also forms a hydrogen bond with one of the phosphonyl oxygen atoms. Other residues (Tyr-177, Gln-408, and Glu-636) interact indirectly with the intermediate analogue by hydrogen-bonding through water molecules. Leu-264 appears to interact with the analogue through Van der Waals contacts, with a terminal methyl carbon making a 3.52 Ä contact distance to the one of the phosphonyl oxygen atoms of the adduct.FIGURE 5Stereo view of the active site environment in the E1-PLThDP structure. The PLThDP is at the interface between the N-terminal and middle domains from different subunits. Residues numbered <470 and >500 are from the N-terminal and middle domains, respectively. The middle domain residues are also shown with brown bonds.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In addition to inducing disorder-to-order transformations, the presence of PLThDP in the active site also induces conformational changes in nearby protein residues that were previously ordered. Relative to the E1-ThDP structure, a conformational rearrangement of protein residues Leu-264, Glu-522, and Tyr-599 occurs in the E1-PLThDP structure. Residue Glu-522 shows a large conformational change as its side chain moved more than 4 Ä. The movement of residues Leu-264 and Tyr-599 and the presence of the phosphonyl oxygen atom close to the thiazolium ring play an important role in the movement of the side chain of Glu-522, which is no longer pointing toward the active site. Although it may not play a direct role in the mechanism of catalysis at this stage, Glu-522 still interacts with the cofactor through water molecules. The key interactions with PLThDP are all likely to be present in the corresponding LThDP intermediate formed from the natural substrate pyruvate, as indicated in Fig. 6, since the majority of good hydrogen bonds can still be formed. Perhaps most surprisingly, analysis of the E1-PLThDP structure revealed a significant distortion in planarity for the C2–C2α bond connecting the substrate analogue to the planar thiazolium ring, as seen in Fig. 4. This out-of-plane distortion implies considerable strain and persists despite incorporation of a planarity restraint during refinement, even after significantly increasing the restraint weight well beyond that normally used. To determine whether this effect is real or simply induced by other possibly incorrect refinement restraints, refinements and pure energy minimizations were carried out with a variety of standard force fields. In all cases, the out-of-plane distortion persists (to varying but significant degrees), provided that any reasonable van der Waals radii are used for the aminopyrimidine N4′ and adduct C2α-hydroxyl O atoms, and in any event, the distortion is required to fit the electron density. The resulting distance between these atoms (2.5 Ä) is reasonable, but it would be unacceptably short (2.2 Ä), producing a strong repulsive force in the absence of the distortion, thus explaining the strained conformation that is observed. Furthermore, since only three essentially rigid groups (coordinates obtained from the highly accurate small molecule PLThDP crystal structure (29Turano A. Furey W. Pletcher J. Sax M. Pike D. Kluger R. J. Am. Chem. Soc. 1982; 104: 3089-3095Crossref Scopus (33) Google Scholar)) are involved, the short contact is dictated by only the thiazolium and aminopyrimidine relative ring orientations and by the fact that the C2α-PO3Me bond is nearly perpendicular to the thiazolium ring plane. The Φt and Φp torsion angles defining the ring orientations, as well as the S–C2–C2α–P torsion angle, are clearly required to fit the strong and unambiguous electron density, and the former are also consistent with those observed in all reported ThDP-containing enzyme structures. Theref