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• W2052206534 abstract "Tagatose-1,6-bisphosphate aldolase (TBPA) is a tetrameric class II aldolase that catalyzes the reversible condensation of dihydroxyacetone phosphate with glyceraldehyde 3-phosphate to produce tagatose 1,6-bisphosphate. The high resolution (1.45 Å) crystal structure of the Escherichia coli enzyme, encoded by the agaY gene, complexed with phosphoglycolohydroxamate (PGH) has been determined. Two subunits comprise the asymmetric unit, and a crystallographic 2-fold axis generates the functional tetramer. A complex network of hydrogen bonds position side chains in the active site that is occupied by two cations. An unusual Na+binding site is created using a π interaction with Tyr183in addition to five oxygen ligands. The catalytic Zn2+ is five-coordinate using three histidine nitrogens and two PGH oxygens. Comparisons of TBPA with the related fructose-1,6-bisphosphate aldolase (FBPA) identifies common features with implications for the mechanism. Because the major product of the condensation catalyzed by the enzymes differs in the chirality at a single position, models of FBPA and TBPA with their cognate bisphosphate products provide insight into chiral discrimination by these aldolases. The TBPA active site is more open on one side than FBPA, and this contributes to a less specific enzyme. The availability of more space and a wider range of aldehyde partners used by TBPA together with the highly specific nature of FBPA suggest that TBPA might be a preferred enzyme to modify for use in biotransformation chemistry. Tagatose-1,6-bisphosphate aldolase (TBPA) is a tetrameric class II aldolase that catalyzes the reversible condensation of dihydroxyacetone phosphate with glyceraldehyde 3-phosphate to produce tagatose 1,6-bisphosphate. The high resolution (1.45 Å) crystal structure of the Escherichia coli enzyme, encoded by the agaY gene, complexed with phosphoglycolohydroxamate (PGH) has been determined. Two subunits comprise the asymmetric unit, and a crystallographic 2-fold axis generates the functional tetramer. A complex network of hydrogen bonds position side chains in the active site that is occupied by two cations. An unusual Na+binding site is created using a π interaction with Tyr183in addition to five oxygen ligands. The catalytic Zn2+ is five-coordinate using three histidine nitrogens and two PGH oxygens. Comparisons of TBPA with the related fructose-1,6-bisphosphate aldolase (FBPA) identifies common features with implications for the mechanism. Because the major product of the condensation catalyzed by the enzymes differs in the chirality at a single position, models of FBPA and TBPA with their cognate bisphosphate products provide insight into chiral discrimination by these aldolases. The TBPA active site is more open on one side than FBPA, and this contributes to a less specific enzyme. The availability of more space and a wider range of aldehyde partners used by TBPA together with the highly specific nature of FBPA suggest that TBPA might be a preferred enzyme to modify for use in biotransformation chemistry. fructose-1,6-bisphosphate aldolase dihydroxyacetone phosphate glyceraldehyde 3-phosphate fructose 1,6-bisphosphate phosphoglycolohydroxamate protonated enzyme tagatose-1,6-bisphosphate aldolase tagatose 1,6-bisphosphate multiwavelength anomalous dispersion root mean square deviation Enzymes are valuable tools for synthetic chemistry, because, under mild conditions, they can provide both high efficiency and optical purity of products (1Takayama S. McGarvey G.J. Wong C.-H. Annu. Rev. Microbiol. 1997; 51: 285-310Crossref PubMed Scopus (83) Google Scholar, 2Wong C.-H. Whitesides G.M. Enzymes in Organic Synthesis. Tetrahedron Organic Chemistry Series Volume 12. Pergamon Press, New York1995Google Scholar). Recombinant DNA technology has increased the availability of useful enzymes, and technologies such as phage display and directed evolution are also being applied toward the discovery of novel bio-catalysts (3Marrs B. Delagrave S. Murphy D. Curr. Opin. Microbiol. 1999; 2: 241-245Crossref PubMed Scopus (68) Google Scholar). The exciting prospect exists that, guided by structural and mechanistic understanding, we can rationally design enzyme activity for the most complex of chemical syntheses. With this long term goal in mind we have undertaken to characterize the structure-activity relationships in metal-dependent aldolases, which are particularly attractive for use in biotransformation chemistry and indeed have already contributed to the synthesis of rare sugars (4von der Osten C.H. Sinskey A.J. Barbas C.F. Pederson R.L. Wang Y.-F. Wong C.-H. J. Am. Chem. Soc. 1989; 111: 3924-3927Crossref Scopus (198) Google Scholar). The most studied aldolases are the fructose-1,6-bisphosphate aldolases (FBPA),1 which participate in two metabolic pathways. FBPA catalyzes the aldol condensation of a ketose, dihydroxyacetone phosphate (glycerone-P or DHAP), and an aldose, glyceraldehyde 3-phosphate (G3P) to form fructose 1,6-bisphosphate (FBP) in gluconeogenesis (see Fig. 1 below). In glycolysis FBPA catalyzes the reverse cleavage. FBP-aldolases are multimers of (α/β)8-barrel subunits and are divided into class I and II enzymes on the basis of mechanism. The type I enzymes utilize a lysine in Schiff base formation during catalysis and are mainly found in higher order organisms as homotetramers of molecular mass around 160 kDa (5Gefflaut T. Blonski C. Perie J. Willson M. Prog. Biophys. Mol. Biol. 1995; 63: 301-340Crossref PubMed Scopus (126) Google Scholar, 6Blom N. Sygusch J. Nat. Struct. Biol. 1997; 4: 36-39Crossref PubMed Scopus (111) Google Scholar). The class II enzymes, found in yeast, bacteria, fungi, and blue-green algae, are most often dimeric and utilize a divalent metal ion, usually Zn2+, in catalysis and, because they are more stable than their class I counterparts, are preferred for use in biotransformation chemistry (4von der Osten C.H. Sinskey A.J. Barbas C.F. Pederson R.L. Wang Y.-F. Wong C.-H. J. Am. Chem. Soc. 1989; 111: 3924-3927Crossref Scopus (198) Google Scholar,7Schoevaart R. van Rantwijk F. Sheldon R.A. J. Org. Chem. 2001; 66: 4559-4562Crossref PubMed Scopus (38) Google Scholar). Structure-activity studies of Escherichia coli class II FBPA have served to delineate some details of the aldol condensation (8Hall D.R. Leonard G.A. Reed C.D. Watt I. Berry A. Hunter W.N. J. Mol. Biol. 1999; 287: 383-394Crossref PubMed Scopus (110) Google Scholar, 9Zgiby S.M. Thomson G.J. Qamar S. Berry A. Eur. J. Biochem. 2000; 267: 1858-1868Crossref PubMed Scopus (50) Google Scholar, 10Plater A. Zgiby S.M. Thomson G.J. Qamar S. Wharton C.W. Berry A. J. Mol. Biol. 1999; 285: 843-855Crossref PubMed Scopus (58) Google Scholar, 11Blom N. Tetreault S. Coulombe R. Sygusch J. Nat. Struct. Biol. 1996; 3: 856-862Crossref PubMed Scopus (81) Google Scholar, 12Qamar S. Marshal K. Berry A. Prot. Sci. 1996; 5: 154-161Crossref PubMed Scopus (32) Google Scholar, 13Cooper S.J. Leonard G.A. McSweeney S.M. Thompson A.W. Naismith J.H. Qamar S. Plater A. Berry A. Hunter W.N. Structure. 1996; 4: 1303-1315Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 14Zgiby S. Plater A.R. Bates M.A. Thomson G.J. Berry A. J. Mol. Biol. 2002; 315: 131-140Crossref PubMed Scopus (42) Google Scholar) (see Fig. 1). Of particular value is the structure of FBPA complexed to phosphoglycolohydroxamate (PGH), which mimics the ene-diolate formed from DHAP (15Collins K.D. J. Biol. Chem. 1974; 249: 136-142Abstract Full Text PDF PubMed Google Scholar), the EH·DHAP(C−) structure (stage II in Fig. 1). Two critical steps in the aldol condensation are: first, the formation of an activated carbon from which a proton is abstracted to produce an enolate and, second, the formation of a new C–C bond. During catalysis, direct coordination of DHAP to Zn2+ aligns the substrate allowing the divalent cation to function as a Lewis acid and to polarize the carbonyl bond of the ketose ready for a reaction that involves three major covalency changes (see Fig. 1). The first covalency change follows abstraction of the1-proS proton of DHAP to produce the ene-diolate, the second is carbon–carbon bond formation to covalently link DHAP C1 with G3P C1 and so form the new C3–C4 bond of a hexose bisphosphate. The substrate oxygen ligands are syn to the enolate bond, and this restricts access of the electrophilic G3P to the Si face of DHAP as the C3–C4 bond is formed. In the third covalency change, a second proton transfer converts the C4 carbonyl to a hydroxyl group and so completes the hexose synthesis. Despite some progress in understanding class II aldolases, there remain aspects of aldolase reactivity that require clarification. We have carried out studies on tagatose-1,6-bisphosphate aldolase (TBPA), a tetrameric enzyme that catalyzes the condensation of DHAP with the natural substrate G3P to form tagatose 1,6-bisphosphate (TBP) (9Zgiby S.M. Thomson G.J. Qamar S. Berry A. Eur. J. Biochem. 2000; 267: 1858-1868Crossref PubMed Scopus (50) Google Scholar, 16Reizer J. Ramseier T.M. Reizer A. Charbit A. Saier M.H.J. Microbiology. 1996; 142: 231-250Crossref PubMed Scopus (63) Google Scholar) a diastereoisomer of FBP (Fig. 1). The reactions catalyzed by TBPA and FBPA (Fig. 1) are similar, and because there is significant conservation of sequence (9Zgiby S.M. Thomson G.J. Qamar S. Berry A. Eur. J. Biochem. 2000; 267: 1858-1868Crossref PubMed Scopus (50) Google Scholar) and structure (this work) a comparison provides insight into class II aldolase mechanism and specificity. The major products of the condensations catalyzed by TBPA and FBPA are distinct in their chirality at C4 (Fig.1). On the basis ofkcat/Km values, FBPA prefers FBP around 1500-fold over TBP so that the overall discrimination between the two substrates is nearly 5 × 105 (9Zgiby S.M. Thomson G.J. Qamar S. Berry A. Eur. J. Biochem. 2000; 267: 1858-1868Crossref PubMed Scopus (50) Google Scholar). FBPA and TBPA, therefore, represent an excellent model system to investigate the molecular basis of chiral discrimination. We now report the high resolution crystal structure of recombinant E. coli TBPA, which encoded by theagaY gene and complexed to PGH, and a detailed comparison with the structure of recombinant E. coli FBPA also complexed with PGH (8Hall D.R. Leonard G.A. Reed C.D. Watt I. Berry A. Hunter W.N. J. Mol. Biol. 1999; 287: 383-394Crossref PubMed Scopus (110) Google Scholar). Although, to date, we have been unable to obtain crystal structures of complexes with a hexose substrate or analogue, we have carried out modeling to generate templates for the TBPA·TBP and FBPA·FBP complexes based on the experimentally derived structures. Established protocols provided the enzyme (9Zgiby S.M. Thomson G.J. Qamar S. Berry A. Eur. J. Biochem. 2000; 267: 1858-1868Crossref PubMed Scopus (50) Google Scholar) and PGH (8Hall D.R. Leonard G.A. Reed C.D. Watt I. Berry A. Hunter W.N. J. Mol. Biol. 1999; 287: 383-394Crossref PubMed Scopus (110) Google Scholar, 15Collins K.D. J. Biol. Chem. 1974; 249: 136-142Abstract Full Text PDF PubMed Google Scholar). The enzyme (6.5 mg ml−1 in 50 mmTris-HCl, pH 7.5) was incubated for 1 h at 4 °C with 20 mm PGH and crystallization achieved in hanging drops of 5 μl of enzyme·PGH complex mixed with 5 μl of reservoir comprising 7–12% (v/v) ethylene glycol, 10 mm ZnCl2, 42 mm sodium cacodylate, pH 6.4. Orthorhombic crystals attained dimensions of 0.20 × 0.25 × 0.30 mm after 3 weeks at 20 °C. The space group is I222 with unit cell lengths ofa = 72.6, b = 100.5, c= 206.7 Å and two TBPA subunits per asymmetric unit. All diffraction measurements were carried out on crystals cryoprotected with ethylene glycol and cooled to −173 °C in a stream of nitrogen gas. Data were first measured in-house using a Rigaku rotating anode (CuKα) and RAXIS IV image plate detector, then at European Synchrotron Radiation Facility stations BM14 and ID14-EH2, for a four-wavelength MAD experiment and the high resolution data, respectively, using MAR charge-coupled device detectors. Wavelengths for the MAD experiment (peak λ1, inflection point λ2, and remote data λ3, λ4) were derived from an x-ray near edge absorption scan (XANES) of the zinc K-edge and selected to maximize the anomalous differences, f″ (λ1), provide the minimum f′ (λ2), and two remote wavelengths (λ3, λ4) to maximize dispersive differences (λ3 − λ2, λ4 − λ2). All data were processed using HKL (17Otwinowski Z. Minor W. Methods Enzymol. 1997; 276A: 307-326Crossref Scopus (38617) Google Scholar). The in-house and high resolution data were scaled together to improve the low resolution terms, and this combined data set was used for refinement (Table I).Table IData and phasing statisticsλ1 (peak)λ2(inflection)λ3 (remote)λ4(remote)Combined in-house/ESRFWavelength (Å)1.28201.28301.12701.0301.5418/0.9330Resolution range29.9–2.2024.5–2.2024.5–2.2024.5–1.9029.8–1.45Measurements127,103127,046177,731204,3351,086,028Unique reflections37,05137,08554,20760,423131,455Coverage overall93.893.889.085.198.4Rsym(%)1-aRsym = Σ‖I − 〈I〉‖/ΣI, where the summation is over all symmetry equivalent reflections.3.2 (11.9)1-bNumbers in parenthesesrefer to a high resolution bin of approximate width 0.1 Å.3.4 (14.5)4.6 (28.0)4.1 (34.8)6.4 (29.2)Ranom(%)1-cRanom = Σ‖I (+) − I(−)‖/Σ(I(+)−I(−)).3.7 (9.3)2.9 (9.9)4.5 (23.9)3.8 (28.4)Phasing power1-dPhasing power is the root-mean-square (‖FH‖/E), whereFH is the calculated heavy-atom structure factor from two Zn2+ positions and E is the residual lack of closure.Accentric/centric0.75 /0.620.78 /0.871.19 /0.791-a Rsym = Σ‖I − 〈I〉‖/ΣI, where the summation is over all symmetry equivalent reflections.1-b Numbers in parenthesesrefer to a high resolution bin of approximate width 0.1 Å.1-c Ranom = Σ‖I (+) − I(−)‖/Σ(I(+)−I(−)).1-d Phasing power is the root-mean-square (‖FH‖/E), whereFH is the calculated heavy-atom structure factor from two Zn2+ positions and E is the residual lack of closure. Open table in a new tab Two Zn2+ positions, identified from an anomalous difference Patterson synthesis, were used to phase the λ2 data to 2.2 Å with a figure-of-merit of 0.54 (18Otwinowski Z. Wolf W. Evans P.R. Leslie A.G.W. Isomorphous Replacement and Anomalous Scattering, Proceedings of the CCP4 Study Weekend. SERC Daresbury Laboratory, Warrington, UK1991: 80-86Google Scholar). Solvent flattening and histogram matching (19Cowtan K. Crystallography. 1994; 31: 34-38Google Scholar, 20Collaborative Computational Project Number 4Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar) followed and raised the overall figure-of-merit to 0.82. Phasing statistics are presented in Table I. Automated procedures (21Anastassis P. Morris R. Lamzin V.S. Nat. Struct. Biol. 1999; 7: 458-463Google Scholar, 22Murshodov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13914) Google Scholar) constructed ∼85% of residues for the two subunits in the asymmetric unit and further rounds of model building, map interpretation (23Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar), and refinement (22Murshodov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13914) Google Scholar) improved this model. It was then refined using the 1.45-Å data set first with rigid body refinement to account for the lack of isomorphism (Riso 0.26) with the MAD data set. Restrained anisotropic refinement without noncrystallographic symmetry restraints was implemented first with CNS (24Brünger 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. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar, 25Sheldrick G.M. Schneider T.R. Methods Enzymol. 1997; 277: 319-343Crossref PubMed Scopus (1892) Google Scholar) then completed using SHELXL with riding hydrogens (25, Table II). The final model consists of residues 2–139 and 151–285 of subunit A and 2–141 and 150–284 of subunit B, two Zn2+ ions, two Na+ ions, 658 waters, and 14 ethylene glycol molecules. Fifteen side chains were modeled in dual conformations, and ten, with poorly defined electron density, were given zero occupancy. There are no Ramachandran outliers, and further details are presented in Table II. The structure and interactions with ligands are well conserved among the two subunits, for example, the 272 Cα atoms common to each overlay with an root mean square deviation of 0.31 Å, and it is therefore appropriate to only detail one active site.Table IIRefinement and model geometry statisticsProtein residues/atoms548/4259PGH atoms/ions/solvents20/2 Zn2+, 2 Na+/658 waters, 14 ethylene glycolsR-work/No. of observations0.13/124,490R-free/No. of observations0.17/6,592Wilson B(Å2)16.2Average isotropic thermal parameters (Å2)Subunit A/B overall18.0/20.6Main chain/side chain16.4/22.4Solvents/PGH/ethylene glycol35.2/17.5/43.4Residues in dual conformations2-aThe single-letter amino acid code, the residue number, and subunit assignment A or B.134A, S39A, R42A, I57A, R91A, V98A, E159A, K7B, I57B, R91B, V98B, R162B, R198B, R272B, V277BResidues for which there is no convincing electron density at the side chain2-aThe single-letter amino acid code, the residue number, and subunit assignment A or B.S151A, K185A, K188A, R257A, S285A, E150B, S151B, K185B, K188B, E216Br.m.s. bond lengths (Å)0.018r.m.s. bond angle associated distances (Å)0.030r.m.s. planarity (Å)0.0252-a The single-letter amino acid code, the residue number, and subunit assignment A or B. Open table in a new tab Molecular docking experiments were performed using AutoDock version 3.0.3 (26Morris G.M. Goodsell D.S. Halliday R.S. Huey R. Hart W.E. Belew R.K. Olson A.J. J. Comput. Chem. 1998; 19: 1639-1662Crossref Scopus (9302) Google Scholar) and GRID (27Goodford P.J. J. Med. Chem. 1985; 28: 849-857Crossref PubMed Scopus (2487) Google Scholar). Receptor models for TBP aldolase and FBP aldolase were derived from the complexes with PGH with all water molecules removed. In addition, for FBPA the side chain of Glu182 was deleted to allow access to Arg331, which has been shown to interact with the G3P phosphate of the substrate (12Qamar S. Marshal K. Berry A. Prot. Sci. 1996; 5: 154-161Crossref PubMed Scopus (32) Google Scholar) and to be consistent with TBPA for which the corresponding residue Glu142 is disordered and missing from the model. Suitable parameters for the Zn2+, Na+, and substrate atoms were derived by reference to literature values (28Erion M.D. Reddy M.R. J. Am. Chem. Soc. 1998; 120: 3295-3304Crossref Scopus (73) Google Scholar) and systematic variation to reproduce the position of PGH observed in the experimentally derived crystal structures. Coordinates for the hexose substrates were prepared using Chem3D (CambridgeSoft) and PRODRG (29van Aalten D.M. Bywater R. Findlay J.B. Hendlich M. Hooft R.W. Vriend G. J. Comput. Aided Mol. Des. 1996; 10: 255-262Crossref PubMed Scopus (579) Google Scholar). The 2–3 bonds (Fig. 1) were fixed, and torsional freedom was allowed in all other 11 torsion angles. Consistent results were achieved using the genetic algorithm with 40 trials and a maximum of 1400 generations. The TBPA subunit comprises 285 residues folded into an (α/β)8barrel carrying three additional helical segments. The secondary structure is mapped onto the amino acid sequence and the three-dimensional arrangement shown in Fig.2. The elements of secondary structure that create the barrel are β1-α2-β2-α4-β3-α5-β4-α6-β5-α7-β6-α8-β7-α9-β8-α10. Helix α1 caps the N-terminal section of the barrel, and α3 comprises two turns of helix linking β2 with α4. Helix α11 is closely associated with α10 and together with the intervening loop forms an “arm” protruding away from the barrel (Figs. 2Band 3A), which provides an important component for subunit·subunit interactions.FIG. 3A, the functional TBPA tetramer with each subunit given a different color and labeledA to D. PGH and cations are included as in Fig.2. The orientation of the subunit shown in Fig. 2 is approximately perpendicular to subunit C in this figure. B, the functional FBPA dimer with subunits A and B in similar orientation to the TBPA subunits A and C. The helices α10 and α11, important for oligomerization, are labeled for both enzymes. FIG. 3, FIG. 4, FIG. 5 were generated using MOLSCRIPT (34Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar) and RASTER-3D (35Merritt E.A. Murphy M.E. P Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 869-873Crossref PubMed Scopus (2859) Google Scholar).View Large Image Figure ViewerDownload (PPT) The TBPA and FBPA monomers have similar dimensions (Fig.3), but a noteworthy difference is that the α10-loop-α11 arm is ∼12 Å shorter in the former. On the basis of a structural alignment the E. coli class II FBPA and the agaY-encoded TBPA enzymes share ∼23% sequence identity (Fig. 2) and 268 Cα atoms from each subunit overlay with an r.m.s.d of 1.56 Å (not shown). However, the quaternary structure differs between the dimeric FBPA and the tetrameric TBPA. The functional TBPA tetramer (subunits A, B, C, and D arranged with 222 point group symmetry) is a rectangular block with approximate dimensions of 92 × 85 × 38 Å (Fig. 3A). The A-B and A-C associations represent two distinct subunit-subunit interfaces. The A-B interface involves the interaction of residues at the N-terminal region of α1 with the turn linking α6 and β5 of an adjacent subunit, resulting in the N-terminal portion of the two (α/β)8 barrels abutting each other in the dimer and the point group symmetry positioning an active site at each corner of the rectangular-shaped tetramer. The two subunits of the TBPA asymmetric unit (A and B) give an arrangement distinct from that observed for functional FBPA. Rather it is the combination of TBPA subunits A and C (or B and D) that forms a dimer similar to FBPA (Fig. 3). This interaction between subunits A and C covers a larger area than the other type of interface (A-B) and is essential to complete the TBPA active site. The A-C interface is formed by the anti-parallel alignment of helices α4 together with interactions from residues at the C-terminal region of the short α3 helix with residues on helices α2 and α4 of the partner subunit. Of particular note is the packing of hydrophobic residues carried on one α10-loop-α11 arm with that of the partner subunit in a manner similar to that observed in FBPA, although this loop is truncated by ∼12 residues in TBPA. Dimer formation by subunits A and C directs the side chain of Arg257 into the active site of the partner subunit in similar fashion to that observed for the C6P binding residue, Arg331 of FBPA (13Cooper S.J. Leonard G.A. McSweeney S.M. Thompson A.W. Naismith J.H. Qamar S. Plater A. Berry A. Hunter W.N. Structure. 1996; 4: 1303-1315Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). The catalytic center is located in a deep, polar cavity at the C-terminal end of the (α/β)8-barrel (Fig.4), and a complex network of hydrogen bonds organize the positions of key functional groups and metal ion ligands. The active site can be divided into a monovalent cation binding site, a divalent cation binding site, the PGH (DHAP) binding site, and the G3P site. TBPA has, like FBPA, a well-defined monovalent cation (Na+) binding site, ∼8.5 Å from the catalytic Zn2+, which serves to create a phosphate binding site and tethers PGH, and by inference the natural substrate DHAP, in the active site. The Na+ coordinates the carbonyl oxygens of four residues (Ala179, Gly181, Gly209, and Ser211) together with a PGH phosphate oxygen. The Na+ O distances fall in the range 2.3–2.6 Å. In FBPA the octahedral Na+ coordination sphere is completed by a water, but in TBPA an uncommon π-cation interaction involving the side chain of Tyr183 is observed (Fig. 4). A similar π-cation interaction involving a Na+·tryptophan association has been observed in lysozyme (30Wouters J. Protein Sci. 1998; 7: 2472-2475Crossref PubMed Scopus (85) Google Scholar), but such a mode of metal binding has, to the best of our knowledge, not been observed before in an enzyme active site. Although the sequence of E. coliFBPA carries a tyrosine (Tyr229) at the same place in the structure as Tyr183 in TBPA, it adopts a different orientation at the surface of FBPA. The differences observed in the FBPA main chain are due to interactions with the extremity of the partner subunit α10-loop-α11 arm, interactions that are absent in TBPA because the arm is truncated as described earlier. The Na+ binding site in TBPA is important for the formation of the PGH/DHAP phosphate binding site and in addition helps to position His180 to coordinate the catalytic Zn2+. The PGH phosphate binds in a site created by strands β7, β8, the loop linking β6 with α8, and the N-terminal region of α10. The latter provides a helix dipole to assist phosphate binding whereas the β8 strand runs close to the inhibitor and imposes steric restrictions at the methylene group of PGH, and by implication C3 on DHAP. In addition to the direct coordination to Na+, the phosphate has two bifurcated hydrogen bond interactions with the main chain amides of Gly181 and Thr233, single hydrogen bonds accepted from the amides of Ser211 and Ala232, the side chain hydroxyls of Ser211 and Thr233 (Fig. 4), and with a water (not shown). The PGH enolate (O1) and hydroxyl (O2) groups chelate the catalytic Zn2+, and a trigonal bipyramidal coordination sphere is completed by three histidines (His83 Nε2, His180 Nε2, and His208 Nδ1, Fig. 4). The metal-ligand distances fall in the range 2.0–2.4 Å. The Zn2+ binding site is well-ordered by a complex network of hydrogen bonding interactions. His83 and His208are held in place by hydrogen bonds donated to the side chain of Asp104 and Glu132 (not shown), respectively. Glu132 also participates in a buried salt bridge with Lys228, which in turn hydrogen bonds to and orients the side chain of Asn230. The side chain of Asn230forms part of the active site floor and donates a weak hydrogen bond of length 3.3 Å to PGH O4. Asn230 is structurally conserved and corresponds to Asn286 in FBPA. In both enzymes the amino group is positioned just under the substrate analogue and serves both to restrict the stereospecificity of the catalytic reaction at DHAP C1 and to assist carbanion formation during catalysis (8Hall D.R. Leonard G.A. Reed C.D. Watt I. Berry A. Hunter W.N. J. Mol. Biol. 1999; 287: 383-394Crossref PubMed Scopus (110) Google Scholar). An asparagine at this position is critical for FBPA, because site-directed mutagenesis of Asn286 to aspartate or alanine greatly reduces kcat by up to 8000-fold (10Plater A. Zgiby S.M. Thomson G.J. Qamar S. Wharton C.W. Berry A. J. Mol. Biol. 1999; 285: 843-855Crossref PubMed Scopus (58) Google Scholar). PGH O1, in addition to coordinating Zn2+, accepts a hydrogen bond from the amide of Gly209 while PGH O2 donates a hydrogen bond to the carboxylate group of Asp82. At this point we note that the residue types and their interactions with each other as described above are conserved between FBPA and TBPA. This strongly implies a conservation of substrate recognition between FBPA and TBPA for DHAP and the enzyme mechanism. Both TBPA and FBPA require deprotonation of DHAP and carbanion formation to produce the unsaturated linkage where the aldol condensation occurs (Fig. 1). There are only a few residues in the active sites that provide a basic group to abstract the acidic1-proS α-H. In FBPA these are Glu182, Asp109, Asp288, and then Asp329from the partner subunit. Site-directed mutagenesis experiments indicate that the aspartates do not abstract the proton from DHAP and implied that Glu182 could be responsible (10Plater A. Zgiby S.M. Thomson G.J. Qamar S. Wharton C.W. Berry A. J. Mol. Biol. 1999; 285: 843-855Crossref PubMed Scopus (58) Google Scholar). Subsequent mutagenesis of Glu182 to alanine and kinetic analysis of the altered protein showed that proton abstraction became rate-limiting, clearly identifying a role in deprotonation (14Zgiby S. Plater A.R. Bates M.A. Thomson G.J. Berry A. J. Mol. Biol. 2002; 315: 131-140Crossref PubMed Scopus (42) Google Scholar). Glu182 is positioned on a flexible loop (8Hall D.R. Leonard G.A. Reed C.D. Watt I. Berry A. Hunter W.N. J. Mol. Biol. 1999; 287: 383-394Crossref PubMed Scopus (110) Google Scholar) about 6.8 Å from the PGH N2 and a conformational alteration would be required to position the side chain where direct abstraction of a proton from DHAP C1 would be feasible. Glu182 of FBPA corresponds to Glu142 in TBPA, which is also positioned on a glycine-rich loop. Due to the lack of reliable electron density, this residue has not been included in the TBPA model and it is presumed to be flexible and disordered. The sequence conservation of this loop in TBPA and FBPA is high (Fig. 2), which implies that Glu142 could adopt a similar position and contribute the same function as Glu182 in FBPA. We would therefore predict that mutagenesis of Glu142 in TBPA would have the same effect as seen for Glu182 in FBPA. An alternative hypothesis, which would still involve Glu182/Glu142, to explain proton abstraction from DHAP C1 involves the use of activated water. In both the FBPA and TBPA·PGH complex structures there is a well-defined water molecule within hydrogen bonding distance of the PGH N2 and correctly positioned to interact with the 1-proS α-H of these class II aldolase·DHAP complexes (Fig. 4). This water therefore represents a potential base for the proton abstraction. In FBPA and TBPA the water is hydrogen bonded to other solvents in the active site and in the former a hydrogen bond network extends up to Glu182 (8Hall D.R. Leonard G.A. Reed C.D. Watt I. Berry A. Hunter W.N. J. Mol. Biol. 1999; 287: 383-394Crossref PubMed Scopus (110) Google Scholar). In the absence of PGH the catalytic Zn2+ of FBPA is five-coordinate, using three histidines and two water molecules (11Blom N. Tetreault S. Coulombe R. Sygusch J. Nat. Struct. Biol. 1996; 3: 856-862Crossref PubMed Scopus (81) Google Scholar). The water ligands are replaced by PGH and, by implication, DHAP during catalysis (8Hall D.R. Leonard G.A. Reed C.D. Watt I. Berry A. Hunter W.N. J. Mol. Biol. 1999; 287: 383-394Crossref PubMed Scopus (110) Google Scholar). The replacement of such cation-binding solvent by hydroxamates is common to other zinc enzyme inhibitor complexes (31Chevrier B. D'orchymont H. Schalk C. Tarnus C. Moras D. Eur. J. Biochem. 1996; 237: 393-398Crossref PubMed Scopus (127) Google Scholar) and a possibility is that the Zn2+ contributes to activating a water. We note that details of a similar proton abstraction step in class I aldolases has proven difficult to explain. However, the recent work of Heine et al. (32Heine A. DeSantis G. Luz J.G. Mitchell M. Wong C.-H. Wilson I.A. Science. 2001; 294: 369-374Crossref PubMed Scopus (263) Google Scholar) has indicated that for d-2-deoxyribose-5-phosphate aldolase a proton relay system activates an active site water, which then mediates proton transfer. Our analysis does not unambiguously explain the details of proton abstraction in the class II aldolase system but does suggest that the potential role of activated water should be investigated. The major differences between the TBPA and FBPA active sites are localized to that region where G3P has to bind, and, although the main chain of the enzymes overlay well, we note two sequence changes that alter side chain contributions to the active sites (Fig.5). Gln59 and Asp288 of FBPA correspond to Ala48 and Ala232, respectively, in TBPA. This results in more space on one side of the TBPA active site and influences the side-chain orientation of a conserved asparagine (Asn35 in FBPA; Asn24 in TBPA). The asparagine Nδ2 groups are structurally conserved when the TBPA and FBPA models are overlaid (not shown) as is the hydrogen bond formed with another conserved residue: Asp109 in FBPA and Asp82 in TBPA. In FBPA, Asn35 Oδ1 is held in place by accepting a hydrogen bond from Gln59 Nε2 whereas Asn35 Nδ2 donates a hydrogen bond to Asp288. In TBPA, however, the loss of functional groups and side-chain atoms results in more space in the TBPA active site. The side chain of TBPA Asn24 adopts a different conformer, and the Oδ1 group alters position with respect to that seen in FBPA. The result is that, although Nδ2 of Asn35 FBPA and Asn25 TBPA occupy the same relative position, the Oδ1 groups occupy different locations in the active site. The complexes with PGH mimic the ene-diolate enzyme structures (stages I through II in Fig. 1) formed from DHAP. We previously modeled the other reactant, G3P into the active site of FBPA on the basis of simple graphical considerations (stage III, 8) but have now used computational chemistry methods to position FBP and TBP in the active sites of their cognate enzymes, in effect to model stage IV (Fig. 1) for each aldolase. Although crude, these models serve to identify possible interactions of relevance to enzyme mechanism and specificity. The lowest energy FBP models in the active site of FBPA, of which one is shown in Fig. 4, placed the C6 phosphate to interact with Arg331 of the partner subunit and Ser61. These residues are conserved in the class II FBP-aldolases, and the model is consistent with the kinetic analysis of mutant enzymes in which these two residues have been altered (9Zgiby S.M. Thomson G.J. Qamar S. Berry A. Eur. J. Biochem. 2000; 267: 1858-1868Crossref PubMed Scopus (50) Google Scholar, 12Qamar S. Marshal K. Berry A. Prot. Sci. 1996; 5: 154-161Crossref PubMed Scopus (32) Google Scholar). The C4 hydroxyl of FBP is positioned about 4 Å from Asn35 and 3 Å from the carboxylate side chain of Asp109. Mutagenesis of the asparagine in FBPA to alanine produced an enzyme with ∼1.5% of the activity of wild-type protein (9Zgiby S.M. Thomson G.J. Qamar S. Berry A. Eur. J. Biochem. 2000; 267: 1858-1868Crossref PubMed Scopus (50) Google Scholar), and the FBPA·FBP model suggests a role in binding the C4 OH. Asp109 has previously been shown to be responsible for the protonation of the incoming C4 carbonyl group (Fig. 1, stage III, 10). The docking of TBP into the active site of TBPA did not produce a set of closely clustered models as observed for the FBPA·FBP combination but indicated that a range of conformations of the G3P component are accessible. This suggests that the TBPA active site allows the substrate more conformational freedom than is the case for FBPA. We present one of the TBP·TBPA models in Fig. 5, which, like the others, suggests that the G3P phosphate interacts with a basic patch, including His26, Lys236, and from the partner subunit Arg257, which corresponds to Arg331 in FBPA discussed above. In addition the phosphate is placed near Thr50, which corresponds to Ser61 in FBPA. His26 and Lys236 are altered in FBPA to Val37 and Gln292, respectively (Fig. 5). The C4 OH group of TBP is directed into the space between the side chains of Ala232 and Asp82 and in position to interact with Asn24. Asp82 corresponds to Asp109 in FBPA and, by analogy with FBPA, we judge it likely that this residue is responsible for the protonation of the C4 carbonyl of during the aldol condensation to produce TBP. An overlap of both enzyme active sites and substrate models (not shown) suggests that there would be a significant steric clash between the G3P of TBP with Asp288 of FBPA in addition to the electrostatic repulsion of anionic groups. Such interactions are likely a contributory discriminating factor in the specificity of FBPA. Superposition of FBP models in the active site of TBPA does not predict any major steric clash. In contrast to FBPA, TBPA displays poor stereochemical control with nearly 10% of the l-erythro configuration being observed and about 90% of the d-threo. In addition, TBPA can utilize glycoaldehyde, l-glyceraldehyde, acetaldehyde, and isobutyraldehyde as the DHAP partner (16Reizer J. Ramseier T.M. Reizer A. Charbit A. Saier M.H.J. Microbiology. 1996; 142: 231-250Crossref PubMed Scopus (63) Google Scholar). This lack of specificity can be explained by the availability of space in that part of the active site where the aldehyde binds. It appears that FBPA has an active site that is complementary to and highly specific for its cognate substrates. TBPA on the other hand is less specific due to the conformational freedom afforded its range of substrates. We conclude that the overall discrimination between the two systems is dominated by the strict specificity of FBPA, and, therefore, TBPA might be the better choice of enzyme to serve as the framework onto which new catalytic activities could be constructed. We thank European Synchrotron Radiation Facility for access and our colleagues for encouragement and excellent support in particular Drs. Graeme Thomson and Shaza Zgiby for help in the preparation of enzyme." @default.
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• W2052206534 title "Structure of Tagatose-1,6-bisphosphate Aldolase" @default.
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