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• W2054120159 abstract "3-Deoxy-d-manno-octulosonate 8-phosphate (KDO8P) synthase catalyzes the condensation of phosphoenolpyruvate (PEP) with arabinose 5-phosphate (A5P) to form KDO8P and inorganic phosphate. KDO8P is the phosphorylated precursor of 3-deoxy-d-manno-octulosonate, an essential sugar of the lipopolysaccharide of Gram-negative bacteria. The crystal structure of the Escherichia coli KDO8P synthase has been determined by multiple wavelength anomalous diffraction and the model has been refined to 2.4 Å (R-factor, 19.9%;R-free, 23.9%). KDO8P synthase is a homotetramer in which each monomer has the fold of a (β/α)8 barrel. On the basis of the features of the active site, PEP and A5P are predicted to bind with their phosphate moieties 13 Å apart such that KDO8P synthesis would proceed via a linear intermediate. A reaction similar to KDO8P synthesis, the condensation of phosphoenolpyruvate, and erythrose 4-phosphate to form 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAH7P), is catalyzed by DAH7P synthase. In the active site of DAH7P synthase the two substrates PEP and erythrose 4-phosphate appear to bind in a configuration similar to that proposed for PEP and A5P in the active site of KDO8P synthase. This observation suggests that KDO8P synthase and DAH7P synthase evolved from a common ancestor and that they adopt the same catalytic strategy. 3-Deoxy-d-manno-octulosonate 8-phosphate (KDO8P) synthase catalyzes the condensation of phosphoenolpyruvate (PEP) with arabinose 5-phosphate (A5P) to form KDO8P and inorganic phosphate. KDO8P is the phosphorylated precursor of 3-deoxy-d-manno-octulosonate, an essential sugar of the lipopolysaccharide of Gram-negative bacteria. The crystal structure of the Escherichia coli KDO8P synthase has been determined by multiple wavelength anomalous diffraction and the model has been refined to 2.4 Å (R-factor, 19.9%;R-free, 23.9%). KDO8P synthase is a homotetramer in which each monomer has the fold of a (β/α)8 barrel. On the basis of the features of the active site, PEP and A5P are predicted to bind with their phosphate moieties 13 Å apart such that KDO8P synthesis would proceed via a linear intermediate. A reaction similar to KDO8P synthesis, the condensation of phosphoenolpyruvate, and erythrose 4-phosphate to form 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAH7P), is catalyzed by DAH7P synthase. In the active site of DAH7P synthase the two substrates PEP and erythrose 4-phosphate appear to bind in a configuration similar to that proposed for PEP and A5P in the active site of KDO8P synthase. This observation suggests that KDO8P synthase and DAH7P synthase evolved from a common ancestor and that they adopt the same catalytic strategy. 3-deoxy-d-manno-octulosonate lipopolysaccharide 3-deoxy-d-manno-octulosonate 8-phosphate phosphoenolpyruvate arabinose 5-phosphate 3-deoxy-d-arabino-heptulosonate 7-phosphate 3-deoxy-d-manno-octulosonate 8-phosphate synthase multiple wavelength anomalous diffraction triose-phosphate isomerase erythrose 4-phosphate 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase helix strand loop 3-Deoxy-d-manno-octulosonate (KDO)1 is an 8-carbon sugar present in the lipopolysaccharide (LPS) of all Gram-negative bacteria (1.Raetz C.R. Annu. Rev. Biochem. 1990; 59: 129-170Crossref PubMed Scopus (1040) Google Scholar). KDO provides a link between lipid A, the membrane embedded moiety of LPS, and the O-antigen, an elongated polysaccharide chain that protrudes from the bacterial outer membrane into the surrounding environment and determines the antigenic specificity of the cell. Although the composition of the O-antigen varies between species and also between strains, the inner core region containing KDO is fairly constant among all Gram-negative bacteria (2.Ray P.H. Kelsey J.E. Bigham E.C. Benedict C.D. Miller T. Am. Chem. Soc. Symp. Ser. 1983; 231: 141-169Crossref Google Scholar). 3-Deoxy-d-manno-octulosonate 8-phosphate synthase (KDO8P synthase, EC 4.1.2.16) plays a key role in the biosynthesis of KDO. This enzyme catalyzes the aldol-type condensation of phosphoenolpyruvate (PEP) with arabinose 5-phosphate (A5P) to form KDO8P (precursor to KDO) and inorganic phosphate (Fig. 1) (3.Levin D.H. Racker E. J. Biol. Chem. 1959; 234: 2532-2539Abstract Full Text PDF PubMed Google Scholar). Dephosphorylation of KDO8P to KDO and synthesis of CMP-KDO (from CTP and KDO) occur prior to insertion of the sugar into LPS (2.Ray P.H. Kelsey J.E. Bigham E.C. Benedict C.D. Miller T. Am. Chem. Soc. Symp. Ser. 1983; 231: 141-169Crossref Google Scholar). Strains ofSalmonella have been isolated with mutations in KDO8P synthase that confer temperature-sensitive growth (4.Rick P.D. Young D.A. J. Bacteriol. 1982; 150: 447-455Crossref PubMed Google Scholar, 5.Rick P.D. Neumeyer B.A. Young D.A. Rev. Infect. Dis. 1984; 6: 455-462Crossref PubMed Scopus (5) Google Scholar). Such strains fail to synthesize KDO at the nonpermissive temperature, which leads to the inhibition of LPS biosynthesis and, as a consequence, to the arrest of cell growth. These studies indicate that KDO8P synthase provides an essential function for bacterial homeostasis. Earlier studies have determined that the reaction of KDO8P synthesis is a sequential process in which the binding of PEP precedes the binding of A5P and the release of inorganic phosphate precedes the release of KDO8P (6.Kohen A. Jakob A. Baasov T. Eur. J. Biochem. 1992; 208: 443-449Crossref PubMed Scopus (58) Google Scholar). The condensation step of the reaction is stereospecific, involving the addition of the si face of C3PEPto the re face of the A5P carbonyl (7.Kohen A. Berkovich R. Belakhov V. Baasov T. Bioorg. Med. Chem. Lett. 1993; 3: 1577-1582Crossref Scopus (59) Google Scholar, 8.Dotson G.D. Nanjappan P. Reily M.D. Woodard R.W. Biochemistry. 1993; 32: 12392-12397Crossref PubMed Scopus (63) Google Scholar). It has also been established that phosphate release occurs by cleavage of the C-O bond of PEP (9.Hedstrom L. Abeles R. Biochem. Biophys. Res. Commun. 1988; 157: 816-820Crossref PubMed Scopus (82) Google Scholar, 10.Dotson G.D. Dua R.K. Clemens J.C. Wooten E.W. Woodard R.W. J. Biol. Chem. 1995; 270: 13698-13705Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) and that the anomeric oxygen of the product KDO8P originates from bulk solvent (10.Dotson G.D. Dua R.K. Clemens J.C. Wooten E.W. Woodard R.W. J. Biol. Chem. 1995; 270: 13698-13705Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). However, the mechanistic details of the condensation reaction remain unclear and very little is known about the residues of the enzyme that are involved in catalysis. As an aid to understanding the mechanism of KDO8P synthase, we have determined the crystal structure of the Escherichia colienzyme at 2.4 Å resolution. 2The structure factor amplitudes of the native data set and the refined coordinates of KDO8P synthase have been deposited in the Protein Data Bank (entry 1D9E).The structure provides a wealth of information on the organization of the active site and on the interactions between the enzyme subunits. A reaction very similar to KDO8P synthesis is the formation of 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAH7P) catalyzed by DAH7P synthase (11.De Leo A.B. Sprinson D.B. Biochem. Biophys. Res. Commun. 1968; 32: 873-877Crossref PubMed Scopus (43) Google Scholar). This reaction is the first step of the shikimate pathway for the biosynthesis of aromatic amino acids. Analysis of the structural and evolutionary relationships between KDO8P synthase and DAH7P synthase provides additional insight into the mechanism of both enzymes and strongly suggests that the syntheses of both KDO8P and DAH7P proceed through the formation of a linear rather than a cyclic intermediate. The E. coli KDO8P synthase (KDO8PS) is encoded by the kdsAgene (12.Woisetschlager M. Hogenauer G. J. Bacteriol. 1986; 168: 437-439Crossref PubMed Google Scholar). The translated monomeric product contains 284 amino acids with a calculated M r of 30,833. KDO8P synthase was purified from E. coli BL21(DE3) harboring thekdsA gene in the plasmid pT7–7 by a modification of the procedure described by Dotson et al. (10.Dotson G.D. Dua R.K. Clemens J.C. Wooten E.W. Woodard R.W. J. Biol. Chem. 1995; 270: 13698-13705Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Ten ml from a small scale growth of the overexpressing strain were transferred to two flasks each containing 1 liter of Luria-Bertani medium supplemented with 100 μg ml−1 ampicillin, and the culture was maintained under shaking at 37 °C until mid-log phase. At this point, expression of KDO8P synthase was induced with 60 μm isopropyl-β-d-thiogalactopyranoside, and the cells were grown for 4 h. Cells were harvested by centrifugation, washed twice with 25 mm potassium phosphate buffer (pH 7.4), and lysed with two passes through a French press. The lysate was centrifuged at 100,000 × g for 1 h, passed through a 0.45-μm filter, and loaded directly onto a DEAE-Sepharose column (200 ml bed volume) equilibrated with 25 mm potassium phosphate (pH 7.4). The column was first washed with three volumes of 25 mm potassium phosphate and then eluted with a two volume gradient from 25 to 130 mmpotassium phosphate. Fractions containing active KDO8P synthase were pooled, and solid ammonium sulfate was added to a final concentration of 0.5 m. The protein solution was then applied to a phenyl-Sepharose column (100-ml bed volume) equilibrated with 25 mm potassium phosphate, 0.5 m ammonium sulfate and eluted with a gradient in one column volume from 25 mmpotassium phosphate, 0.5 m ammonium sulfate to 25 mm potassium phosphate. Fractions containing the active enzyme were pooled and concentrated by pressure filtration. The concentrated enzyme (80 mg ml−1) in 25 mmpotassium phosphate was flash frozen and stored in liquid nitrogen in small aliquots until crystallization. Crystallization was performed by vapor diffusion in hanging drops. Cubic crystals (space groupI23, a = 228.6 Å) were obtained from drops containing 400 mm potassium phosphate, pH 7.5, 1.4m ammonium sulfate, 6% ethylene glycol, 4 mmPEP, 4 mm A5P, and 30 mg ml−1 KDO8PS, equilibrated at 23 °C against 400 mm potassium phosphate, 6% ethylene glycol, and 1.7–1.9 m ammonium sulfate. Crystals were harvested and maintained in a cryo-protectant holding solution containing 400 mm potassium phosphate, pH 7.5, 2.4 m ammonium sulfate, 6% ethylene glycol, 10% glycerol. KDO8PS activity was determined by measuring the amount of KDO8P produced using the periodate-thiobarbituric acid assay (13.Ray P.H. J. Bacteriol. 1980; 141: 635-644Crossref PubMed Google Scholar). Protein concentration was determined by the method of Lowry (14.Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). A native data set was collected at 100 K with a Raxis IV image plate detector at the CuKα wavelength. The structure determination of the enzyme employed the anomalous diffraction at multiple wavelengths (MAD) of a mercury derivative (Table I) obtained by soaking crystals for 24 h in a holding solution containing 9 mm mercurochrome (2′,7′-dibromo-4′-[hydroxymercurio]-fluorescein) and sodium sulfate in place of ammonium sulfate. Anomalous diffraction at the absorption edge of mercury and at a remote wavelength were collected at beamline X12C, Brookhaven National Laboratories, with a charge-coupled device detector. Oscillation data were processed with HKL (15.Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38573) Google Scholar). Three heavy atom sites were identified with SOLVE (16.Terwilliger T.C. Berendzen J. Acta Crystallogr. Sec. D. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar), which was also used for MAD phasing in combination with SHARP (17.La Fortelle E.D. Irwin J.J. Bricogne G. Crystallogr. Comput. 1997; 7: 1-9Google Scholar). Density modification and 2-fold noncrystallographic symmetry averaging with DM (18.Collaborative Computational Project Number 4Acta Crystallogr. Sec. D. 1994; 50: 760-763Crossref PubMed Scopus (19770) Google Scholar) and SOLOMON (18.Collaborative Computational Project Number 4Acta Crystallogr. Sec. D. 1994; 50: 760-763Crossref PubMed Scopus (19770) Google Scholar) gave an interpretable map at 3.2 Å. Phases were improved and extended stepwise (0.15 Å increments) to 2.4 Å (resolution of the native data set). The model for subunits A and B was fit into the density using O (19.Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sec. A. 1991; 47: 110-119Crossref PubMed Scopus (13011) Google Scholar). Subunits C and D were generated by application of the local symmetry. Model refinement was carried out with CNS version 0.5 (20.Brunger 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 M. Simonson T. Warren G.L. Acta Crystallogr. D. 1998; 54: 905-921Crossref PubMed Scopus (16967) Google Scholar) using cross-validated maximum likelihood as the target function (21.Adams P.D. Pannu N.S. Read R.J. Brunger A.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5018-5023Crossref PubMed Scopus (383) Google Scholar). Solvent molecules were added only during the final stages of refinement after the protein model had stabilized. Residues 206–217 (L7) and 246–251 (L8) of subunits A, B, and C, and residues 206–217 and 245–252 of subunit D could not be identified unambiguously and were not included in the final model. The global and local validity of the model were assessed with the programs SFCHECK (22.Vaguine A.A. Richelle J. Wodak S.J. Acta Crystallogr. Sec. D. 1999; 55: 191-205Crossref PubMed Scopus (859) Google Scholar) and DDQ (23.van den Akker F. Hol W.G. Acta Crystallogr. Sec. D. 1999; 55: 206-218Crossref PubMed Scopus (48) Google Scholar).Table IData collection, MAD phasing, and model refinement statisticsNativeMercurochromeData collection Wavelength (Å)1.54180.970002 (remote)1.007524 (edge) Resolution range (Å)23–2.429–3.230–3.2 Measurements1,582,7161,279,7951,261,629 Unique reflections76,68433,32433,252 <Redundancy>20.638.437.9 Completeness (%)aValues in parentheses refer to the highest resolution bin (0.15 Å wide).99.3 (94.6)99.8 (100.0)99.8 (99.9) <I>/ς<I>22.3 (2.3)27.5 (6.8)26.1 (5.9) R merge(%)bRmerge = ΣhΣi‖I(h)i − <I(h)>‖/ΣhΣi I(h)i, where I(h)i is the ith measurement.8.6 (56.4)13.7 (55.8)14.5 (59.3)Phasing statistics (29–3.2 Å) Mean figure of merit0.52 (0.48) R Cullis (dispersive) (%)cRCullis(dispersive) = <E>/<‖F remote −F edge‖>, where E is the phase-integrated lack of closure and F remote andF edge are the structure factors at the remote and edge wavelength, respectively. R Cullis(anomalous) is the ratio between the anomalous lack of closure and the anomalous difference.47.8 (53.7) R Cullis(anomalous) (%)83.8 (97.7)93.6 (99.4) Phasing power (dispersive)dPhasing power = <‖FH‖/E>, where F H is the calculated heavy atom structure factor. Rcryst = Σ‖Fobs − Fcalc‖/Σ‖Fobs‖. R freewas calculated on 10% of the data omitted from refinement. Stereochemistry was assessed with PROCHECK (18).2.26 (1.13) Phasing power (anomalous)1.54 (0.77)0.83 (0.43)Refinement statistics (23–2.4 Å, no ς cutoff) Rcryst(%)19.9 Rfree (%)23.9 Amino acids1062 Water molecules446 <B> (Å2) protein49.5 <B> (Å2) waters49.9 rmsd bond lengths (Å)0.012 rmsd bond angles (deg)1.574 rmsd impropers (deg)1.012 ϕψ angles (%)Most favored90.6Allowed9.4a Values in parentheses refer to the highest resolution bin (0.15 Å wide).b Rmerge = ΣhΣi‖I(h)i − <I(h)>‖/ΣhΣi I(h)i, where I(h)i is the ith measurement.c RCullis(dispersive) = <E>/<‖F remote −F edge‖>, where E is the phase-integrated lack of closure and F remote andF edge are the structure factors at the remote and edge wavelength, respectively. R Cullis(anomalous) is the ratio between the anomalous lack of closure and the anomalous difference.d Phasing power = <‖FH‖/E>, where F H is the calculated heavy atom structure factor. Rcryst = Σ‖Fobs − Fcalc‖/Σ‖Fobs‖. R freewas calculated on 10% of the data omitted from refinement. Stereochemistry was assessed with PROCHECK (18.Collaborative Computational Project Number 4Acta Crystallogr. Sec. D. 1994; 50: 760-763Crossref PubMed Scopus (19770) Google Scholar). Open table in a new tab Multiple sequence alignments were performed with the program ClustalW (24.Higgins D.G. Sharp P.M. Gene (Amst.). 1988; 73: 237-244Crossref PubMed Scopus (2878) Google Scholar). Structure superposition and structure-based sequence alignment were carried out using the program LSQMAN (25.Kleywegt G.J. Jones T.A. Methods Enzymol. 1997; 277: 525-545Crossref PubMed Scopus (303) Google Scholar). The asymmetric unit of the crystal contains a homotetramer of KDO8P synthase (subunits ABCD) with 222 local symmetry (three 2-fold axes intersecting in the tetramer center) (Fig.2); each monomer has the fold of a typical (β/α)8 barrel (eight-stranded parallel β-barrel surrounded by eight helices) as observed in the structure of triose-phosphate isomerase (TIM) (26.Banner D.W. Bloomer A.C. Petsko G.A. Phillips D.C. Pogson C.I. Wilson I.A. Corran P.H. Furth A.J. Milman J.D. Offord R.E. Priddle J.D. Waley S.G. Nature. 1975; 255: 609-614Crossref PubMed Scopus (598) Google Scholar). The secondary structure elements of the E. coli KDO8P synthase (284 residues) and TIM from various sources (247–250 residues) superimpose well (not shown) with the main differences occurring at the N terminus, where KDO8P synthase has an additional β-hairpin that seals the N-terminal end of the barrel, and at the C terminus, where helix H8 of KDO8P synthase has a random coil extension (Fig. 2). Despite the significant structural kinship between KDO8P synthase and TIM, neither a direct sequence comparison nor a structure based alignment reveals any sequence similarity between the two proteins. Previous biochemical studies indicated that the E. coliKDO8P synthase is a trimer of identical subunits (13.Ray P.H. J. Bacteriol. 1980; 141: 635-644Crossref PubMed Google Scholar). However, in the cubic crystals of KDO8P synthase there are no intersubunit contacts along the directions of the crystallographic 3-fold axes that would support a trimeric architecture. A tetramer is also contained in the asymmetric unit of a different crystal form of KDO8P synthase belonging to a monoclinic space group. 3S. Radaev, P. Dastidar, M. Patel, R. W. Woodard, and D. L. Gatti, manuscript in preparation.Together these observations suggest that the tetramer probably represents the native form of the enzyme. The four monomers of KDO8P synthase are very similar with root mean square deviations not exceeding 1.0 Å for all atoms and 0.5 Å for Cα atoms in all pairwise comparisons. The contact surface between subunits A and B (or C and D) is characterized by both polar and hydrophobic interactions occurring primarily between helices H6, H7, and H8 of one subunit and the same helices of the other subunit (Fig. 2). Two loops play a key role in completing the assembly of the tetramer: loop L2 (residues 58–72) of subunit A contacts helices H4 and H5 of subunit C and loop L6 (residues 170–182) of subunit A contacts the same loop of subunit C at the center of the tetramer. Also, the C-terminal coil extension of each subunit extends over helices H7 and H8 of the neighboring subunit (Fig. 2). Because of the 222 symmetry of the tetramer, the interactions described above (A to C) occur again between the same subunits in reverse (C to A) and between the other two subunits (B to D and D to B). An interesting feature of KDO8P synthase crystals is that the three-dimensional lattice is sparsely populated (70% solvent content) with only subunits A, B, and C involved in crystal contacts. This characteristic packing may be responsible for the fact that the temperature factors of subunit D are about 30% higher than those of the other three subunits of the enzyme. Attempts were made to visualize the substrates PEP and A5P by incubating crystals of KDO8P synthase in the presence of a large excess (4 × 104-fold) of either of these compounds over their K m values ( KmPEP = 6 μm; KmA5P = 30 μm; Ref.6.Kohen A. Jakob A. Baasov T. Eur. J. Biochem. 1992; 208: 443-449Crossref PubMed Scopus (58) Google Scholar). Unfortunately, neither PEP nor A5P were visible under these conditions, probably because of the high ionic strength of the holding solution (2.4 m ammonium sulfate). Attempts to transfer crystals of KDO8P synthase to a low ionic strength holding solution were not successful. Although the active site of KDO8P synthase could not be identified directly, its location could be derived by comparison with the structures of other enzymes that adopt a TIM barrel topology. In these enzymes the active site is always located in a cavity at the C-terminal end of the barrel (27.Babbitt P.C. Gerlt J.A. J. Biol. Chem. 1997; 272: 30591-30594Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). A similar motif is also observed in KDO8P synthase, where an elongated depression at the interface between subunits opens into a deeper cavity that is likely to represent the active site. Two cavities are visible on each face of the tetramer, one between subunit A and C and one between subunit B and D (Figs. 2 and3 A). Most of the residues of the putative active site are contributed by the C-terminal end of the barrel and by the loops of one subunit (Fig. 3 A, yellow shading) with a small contribution originating from helices H4 and H5, and loops L4 and L5 of another subunit (Fig. 3 A,cyan shading). A sulfate ion (SO4–1), stabilized by salt bridges to the side chains of Lys-138 and Arg-168 and by a hydrogen bond to the backbone amide of Ala-116, is located in the most recessed part of the active site cavity (Fig. 3 B). The pattern of noncovalent interactions around this ion is very similar to that observed in the structure of yeast enolase from crystals stabilized in ammonium sulfate, in which a sulfate ion is found at the location of the phosphate moiety of PEP (28.Stec B. Lebioda L. J. Mol. Biol. 1990; 211: 235-248Crossref PubMed Scopus (88) Google Scholar). Because of the high concentration of ammonium sulfate at which the crystals are maintained, it is possible that a sulfate ion also substitutes for PEP in the active site of KDO8P synthase. Additional evidence that the position of SO4–1 represents the binding site of the phosphate moiety of PEP derives from the observation that PEP, but not A5P, protects His-202 (whose Nδ is 4.7 Å from one of the oxygen atoms of SO4–1, Fig.3 B) from chemical modification by diethylpyrocarbonate (29.Sheflyan G.Y. Duewel H.S. Chen G. Woodard R.W. Biochemistry. 1999; 38: 14320-14329Crossref PubMed Scopus (14) Google Scholar). A second sulfate ion (SO4–2) is located approximately 13 Å from SO4–1, in a raised position above the opening of the barrel and beneath the very long L2 loop (residues 58–72) (Fig. 3 B). This sulfate ion is stabilized by a salt bridge to the side chain of Arg-63 and by hydrogen bonds to both the side chain and the backbone of Ser-64. The distance between the aldehyde carbon (C1) and the phosphate moiety of A5P is approximately 7.5 Å, and the distance between C3 and the phosphate group of PEP is approximately 3.5 Å. The sum of these distances (11 Å) matches closely the distance of 13 Å between SO4–1 and SO4–2, which suggests that PEP and A5P may bind with their phosphate moieties occupying the sites of these sulfate ions and with C3PEP in close proximity of C1A5P. The proposed positions of PEP and A5P, with PEP located more deeply in the active site and A5P closer to the opening, are consistent with the observation that the synthesis of KDO8P is an ordered reaction in which the binding of PEP precedes the binding of A5P (6.Kohen A. Jakob A. Baasov T. Eur. J. Biochem. 1992; 208: 443-449Crossref PubMed Scopus (58) Google Scholar). In subunit B and C only, a third sulfate ion (SO4–3) is bound near the outer rim of the active site cavity, in a region that includes contributions from L1 and H1 (Figs. 2 and 3). This sulfate ion is stabilized by a salt bridge to Arg-31 and possibly by the positive charge of the N-terminal end of helix H1. The fact that only two of four potential SO4–3 sites are occupied, despite equal accessibility of Arg-31 in all four subunits of the enzyme, suggests that the initial binding of sulfate to one face of KDO8P synthase (subunits B and C in Figs. 2 and 3 A) introduces an asymmetry in the tetramer that may affect the binding of sulfate to the other face. The physiological function of the SO4–3 site remains uncertain. However, it is of interest that inorganic phosphate is a noncompetitive inhibitor of theE. coli KDO8P synthase (6.Kohen A. Jakob A. Baasov T. Eur. J. Biochem. 1992; 208: 443-449Crossref PubMed Scopus (58) Google Scholar). Thus, the SO4–3 site identified in the x-ray structure of the enzyme may correspond to the inhibitory phosphate binding site predicted from kinetic measurements. In this context, it is of note that the binding of sulfate at the SO4–3 site of subunit B and C coincides with several small structural changes in the side chains of Asp-32, Arg-36, Glu-245, and Pro-252, which appear to propagate from Arg-31 in the direction of the active site cavity. A reaction similar to KDO8P synthesis constitutes the first step in the metabolic pathway for the biosynthesis of aromatic amino acids. This reaction is the formation of DAH7P from erythrose 4-phosphate (E4P) and PEP, which is catalyzed by DAH7P synthase (DAH7PS, EC 4.1.2.15) (11.De Leo A.B. Sprinson D.B. Biochem. Biophys. Res. Commun. 1968; 32: 873-877Crossref PubMed Scopus (43) Google Scholar). Bacteria and fungi both express several isoforms of DAH7P synthase that are characterized primarily by differences in their allosteric regulators. Although there is no obvious sequence similarity between the E. coli KDO8P synthase and the three isoforms of the E. coli DAH7P synthase, there is sequence similarity between theE. coli KDO8P synthase and the DAH7P synthases fromChlamydia thrachomatis, Bacillus subtilis (strainMarburg168), and Aeropyrum pernix. A multiple alignment of KDO8P and DAH7P synthase sequences reveals that most of the residues involved in the architecture of the active site (see above) are conserved in both groups of enzymes (Fig.4). In particular, all the residues directly involved in the binding of SO4–1 and SO4–2 are highly conserved, suggesting that the binding modes of PEP and A5P or PEP and E4P in the active sites of these homologous enzymes are similar. In contrast, Arg-31, which is the residue primarily responsible for the stabilization of SO4–3, is present only in a subset of all the sequences, indicating that the SO4–3 site may not be a universal feature of KDO8P and DAH7P synthases. As previously mentioned, analysis of the amino acid sequences of theE. coli KDO8P synthase (284 residues) and of the E. coli isoforms of DAH7P synthase (∼350 residues each) does not reveal any obvious similarities. Furthermore, the E. coliDAH7P synthase isozymes require a metal ion for activity (30.Stephens C.M. Bauerle R. J. Biol. Chem. 1991; 266: 20810-20817Abstract Full Text PDF PubMed Google Scholar), whereas no metals can be detected by atomic absorption or UV-visible spectroscopy in active preparations of the E. coli KDO8P synthase (data not shown) nor is any metal observed in the x-ray structure of the enzyme. These differences might be interpreted as representative of the fact that, although the two enzymes catalyze very similar reactions, they are not evolutionarily related. Surprisingly, the recently reported crystal structure of the E. coliphenylalanine-regulated DAH7P synthase (31.Shumilin I.A. Kretsinger R.H. Bauerle R.H. Structure. 1999; 7: 865-875Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar) shows that both the tertiary and quaternary structures of this enzyme are remarkably similar to those of KDO8P synthase. Like KDO8P synthase, DAH7P synthase is a homotetramer with 222 symmetry. A superposition of the monomers of the E. coli KDO8P and DAH7P synthases is shown in Fig.5. The core structure, represented by the elliptical (β/α)8 barrel (colored in yellowfor KDO8PS and blue for DAH7PS), is almost identical in the two enzymes. However, several important structural features are unique to each protein. The N terminus of DAH7P synthase contains an extension of 50 amino acids that includes one short β-strand and two helices (colored in cyan in Fig. 5). The second of the two helices seals the N-terminal end of the barrel and thus occupies a position corresponding to that of the N-terminal β-hairpin in KDO8P synthase (colored in orange in Fig. 5, see also Fig. 2). The L2 loop, which in KDO8PS is involved in the binding of SO4–2, is substantially longer and convoluted in DAH7PS (colored in green in Fig.5). In DAH7P synthase the connection between helix H5 and strand S6 is provided by a β-hairpin (strands S6a and S6b, colored inpink in Fig. 5) that extends outside of the barrel. This hairpin forms a three-stranded antiparallel β-sheet with the short N-terminal strand of a neighboring monomer and thus is important for the assembly of the DAH7PS tetramer. Finally, the L8 loop (colored inmagenta in Fig. 5) is longer in DAH7PS than in KDO8PS; however, in both enzymes this loop is partially disordered. A comparison of the binding interactions of ions and substrates in the active sites of KDO8P and DAH7P synthases provides insight into the mechanism and evolution of these enzymes. KDO8PS and DAH7PS adhere almost perfectly to the canonical characteristics of enzymes with a TIM barrel topology (27.Babbitt P.C. Gerlt J.A. J. Biol. Chem. 1997; 272: 30591-30594Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). Their active site is locate" @default.
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• W2054120159 title "Structure and Mechanism of 3-Deoxy-d-manno-octulosonate 8-Phosphate Synthase" @default.
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