Structure of 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase from Pseudomonas putida.

2-Keto-3-deoxy-6-phosphogluconate (KDPG) aldolase from Pseudomonas putida is a key enzyme in the Entner-Doudoroff pathway which catalyses the cleavage of KDPG via a class I Schiff-base mechanism. The crystal structure of this enzyme has been refined to a crystallographic residual R = 17.1% (R(free) = 21.4%). The N-terminal helix caps one side of the torus of the (betaalpha)(8)-barrel and the active site is located on the opposite, carboxylic side of the barrel. The Schiff-base-forming Lys145 is coordinated by a sulfate (or phosphate) ion and two solvent water molecules. The interactions that stabilize the trimer are predominantly hydrophobic, with the exception of the cyclically permuted bonds formed between Glu132 OE1 of one molecule and Thr129 OG1 of a symmetry-equivalent molecule. Except for the N-terminal helix, the structure of KDPG aldolase from P. putida closely resembles the structure of the homologous enzyme from Escherichia coli.

Human thymidylate synthase is in the closed conformation when complexed with dUMP and raltitrexed, an antifolate drug.

Thymidylate synthase (TS) is a major target in the chemotherapy of colorectal cancer and some other neoplasms while raltitrexed (Tomudex, ZD1694) is an antifolate inhibitor of TS approved for clinical use in several European countries. The crystal structure of the complex between recombinant human TS, dUMP, and raltitrexed has been determined at 1.9 A resolution. In contrast to the situation observed in the analogous complex of the rat TS, the enzyme is in the closed conformation and a covalent bond between the catalytic Cys 195 and dUMP is present in both subunits. This mode of ligand binding is similar to that of the analogous complex of the Escherichia coli enzyme. The only major differences observed are a direct hydrogen bond between His 196 and the O4 atom of dUMP and repositioning of the side chain of Tyr 94 by about 2 A. The thiophene ring of the drug is disordered between two parallel positions.

Structure of human thymidylate synthase suggests advantages of chemotherapy with noncompetitive inhibitors.

Thymidylate synthase (TS) is a major target in the chemotherapy of colorectal cancer and some other neoplasms. The emergence of resistance to the treatment is often related to the increased levels of TS in cancer cells, which have been linked to the elimination of TS binding to its own mRNA upon drug binding, a feedback regulatory mechanism, and/or to the increased stability to intracellular degradation of TS.drug complexes (versus unliganded TS). The active site loop of human TS (hTS) has a unique conformation resulted from a rotation by 180 degrees relative to its orientation in bacterial TSs. In this conformation, the enzyme must be inactive, because the catalytic cysteine is no longer positioned in the ligand-binding pocket. The ordered solvent structure obtained from high resolution crystallographic data (2.0 A) suggests that the inactive loop conformation promotes mRNA binding and intracellular degradation of the enzyme. This hypothesis is supported by fluorescence studies, which indicate that in solution both active and inactive forms of hTS are present. The binding of phosphate ion shifts the equilibrium toward the inactive conformation; subsequent dUMP binding reverses the equilibrium toward the active form. Thus, TS inhibition via stabilization of the inactive conformation should lead to less resistance than is observed with presently used drugs, which are analogs of its substrates, dUMP and CH(2)H(4)folate, and bind in the active site, promoting the active conformation. The presence of an extension at the N terminus of native hTS has no significant effect on kinetic properties or crystal structure.

Cation binding and thermostability of FTHFS monovalent cation binding sites and thermostability of N10-formyltetrahydrofolate synthetase from Moorella thermoacetica.

Formyltetrahydrofolate synthetase (FTHFS) from the thermophilic homoacetogen, Moorella thermoacetica, has an optimum temperature for activity of 55-60 degrees C and requires monovalent cations for both optimal activity and stabilization of tetrameric structure at higher temperatures. The crystal structures of complexes of FTHFS with cesium and potassium ions were examined and monovalent cation binding positions identified. Unexpectedly, NH(4)(+) and K(+), both of which are strongly activating ions, bind at a different site than a moderately activating ion, Cs(+), does. Neither binding site is located in the active site. The sites are 7 A apart, but in each of them, the side chain of Glu 98, which is conserved in all known bacterial FTHFS sequences, participates in metal ion binding. Other ligands in the Cs(+) binding site are four oxygen atoms of main chain carbonyls and water molecules. The K(+) and NH(4)(+) binding site includes the carboxylate of Asp132 in addition to Glu98. Mutant FTHFS's (E98Q, E98D, and E98S) were obtained and analyzed using differential scanning calorimetry to examine the effect of these mutations on the thermostability of the enzyme with and without added K(+) ions. The addition of 0.2 M K(+) ions to the wild-type enzyme resulted in a 10 degrees C increase in the thermal denaturation temperature. No significant increase was observed in E98D or E98S. The lack of a significant effect of monovalent cations on the stability of E98D and E98S indicates that this alteration of the binding site eliminates cation binding. The thermal denaturation temperature of E98Q was 3 degrees C higher than that of the wild-type enzyme in the absence of the cation, indicating that the removal of the unbalanced, buried charge of Glu98 stabilizes the enzyme. These results confirm that Glu98 is a crucial residue in the interaction of monovalent cations with FTHFS.

The H159A mutant of yeast enolase 1 has significant activity.

The function of His159 in the enolase mechanism is disputed. Recently, Vinarov and Nowak (Biochemistry (1999) 38, 12138-12149) prepared the H159A mutant of yeast enolase 1 and expressed this in Escherichia coli. They reported minimal (ca. 0.01% of the native value) activity, though the protein appeared to be correctly folded, according to its CD spectrum, tryptophan fluorescence, and binding of metal ion and substrate. We prepared H159A enolase using a multicopy plasmid and expressed the enzyme in yeast. Our preparations of H159A enolase have 0.2-0.4% of the native activity under standard assay conditions and are further activated by Mg(2+) concentrations above 1 mM to 1-1.5% of the native activity. Native enolase 1 (and enolase 2) are inhibited by such Mg(2+) concentrations. It is possible that His159 is necessary for correct folding of the enzyme and that expression in E. coli leads to largely misfolded protein.

Catalytic cysteine of thymidylate synthase is activated upon substrate binding.

The role of Ser 167 of Escherichia coli thymidylate synthase (TS) in catalysis has been characterized by kinetic and crystallographic studies. Position 167 variants including S167A, S167N, S167D, S167C, S167G, S167L, S167T, and S167V were generated by site-directed mutagenesis. Only S167A, S167G, S167T, and S167C complemented the growth of thymidine auxotrophs of E. coli in medium lacking thymidine. Steady-state kinetic analysis revealed that mutant enzymes exhibited k(cat) values 1.1-95-fold lower than that of the wild-type enzyme. Relative to wild-type TS, K(m) values of the mutant enzymes for 2'-deoxyuridylate (dUMP) were 5-90 times higher, while K(m) values for 5,10-methylenetetrahydrofolate (CH(2)H(4)folate) were 1.5-16-fold higher. The rate of dehalogenation of 5-bromo-2'-deoxyuridine 5'-monophosphate (BrdUMP), a reaction catalyzed by TS that does not require CH(2)H(4)folate as cosubstrate, by mutant TSs was analyzed and showed that only S167A and S167G catalyzed the dehalogenation reaction and values of k(cat)/K(m) for the mutant enzymes were decreased by 10- and 3000-fold, respectively. Analysis of pre-steady-state kinetics of ternary complex formation revealed that the productive binding of CH(2)H(4)folate is weaker to mutant TSs than to the wild-type enzyme. Chemical transformation constants (k(chem)) for the mutant enzymes were lower by 1.1-6.0-fold relative to the wild-type enzyme. S167A, S167T, and S167C crystallized in the I2(1)3 space group and scattered X-rays to either 1.7 A (S167A and S167T) or 2.6 A (S167C). The high-resolution data sets were refined to a R(crys) of 19.9%. In the crystals some cysteine residues were derivatized with 2-mercaptoethanol to form S,S-(2-hydroxyethyl)thiocysteine. The pattern of derivatization indicates that in the absence of bound substrate the catalytic cysteine is not more reactive than other cysteines. It is proposed that the catalytic cysteine is activated by substrate binding by a proton-transfer mechanism in which the phosphate group of the nucleotide neutralizes the charge of Arg 126', facilitating the transfer of a proton from the catalytic cysteine to a His 207-Asp 205 diad via a system of ordered water molecules.

The crystal structure and amino acid sequence of dehaloperoxidase from Amphitrite ornata indicate common ancestry with globins.

The full-length, protein coding sequence for dehaloperoxidase was obtained using a reverse genetic approach and a cDNA library from marine worm Amphitrite ornata. The crystal structure of the dehaloperoxidase (DHP) was determined by the multiple isomorphous replacement method and was refined at 1.8-A resolution. The enzyme fold is that of the globin family and, together with the amino acid sequence information, indicates that the enzyme evolved from an ancient oxygen carrier. The peroxidase activity of DHP arose mainly through changes in the positions of the proximal and distal histidines relative to those seen in globins. The structure of a complex of DHP with 4-iodophenol is also reported, and it shows that in contrast to larger heme peroxidases DHP binds organic substrates in the distal cavity. The binding is facilitated by the histidine swinging in and out of the cavity. The modeled position of the oxygen atom bound to the heme suggests that the enzymatic reaction proceeds via direct attack of the oxygen atom on the carbon atom bound to the halogen atom.

The crystal structure of N(10)-formyltetrahydrofolate synthetase from Moorella thermoacetica.

The structure was solved at 2.5 A resolution using multiwavelength anomalous dispersion (MAD) scattering by Se-Met residues. The subunit of N(10)-formyltetrahydrofolate synthetase is composed of three domains organized around three mixed beta-sheets. There are two cavities between adjacent domains. One of them was identified as the nucleotide binding site by homology modeling. The large domain contains a seven-stranded beta-sheet surrounded by helices on both sides. The second domain contains a five-stranded beta-sheet with two alpha-helices packed on one side while the other two are a wall of the active site cavity. The third domain contains a four-stranded beta-sheet forming a half-barrel. The concave side is covered by two helices while the convex side is another wall of the large cavity. Arg 97 is likely involved in formyl phosphate binding. The tetrameric molecule is relatively flat with the shape of the letter X, and the active sites are located at the end of the subunits far from the subunit interface.

Reactions of Pseudomonas 7A glutaminase-asparaginase with diazo analogues of glutamine and asparagine result in unexpected covalent inhibitions and suggests an unusual catalytic triad Thr-Tyr-Glu.

Pseudomonas 7A glutaminase-asparaginase (PGA) catalyzes the hydrolysis of D and L isomers of glutamine and asparagine. Crystals of PGA were reacted with diazo analogues of glutamine (6-diazo-5-oxo-L-norleucine, DON) and asparagine (5-diazo-4-oxo-L-norvaline, DONV), which are known inhibitors of the enzyme. The derivatized crystals remained isomorphous to native PGA crystals. Their structures were refined to crystallographic R = 0.20 and R(free) = 0.24 for PGA-DON and R = 0.19 and R = 0.23 for PGA-DONV. Difference Fourier electron density maps clearly showed that both DON and DONV inactivate PGA through covalent inhibition. Continuous electron density connecting the inhibitor to both Thr20 and Tyr34 of the flexible loop was observed providing strong evidence that Thr20 is the primary catalytic nucleophile and that Tyr34 plays an important role in catalysis as well. The unexpected covalent binding observed in the PGA-DON and PGA-DONV complexes shows that a secondary reaction involving the formation of a Tyr34-inhibitor bond takes place with concomitant inactivation of PGA. The predicted covalent linkage is not seen, however, suggesting an alternative method of inhibition not yet seen for these diazo analogues. These surprising results give insight as to the role of the flexible loop Thr and Tyr in the catalytic mechanism.

Drug-resistant variants of Escherichia coli thymidylate synthase: effects of substitutions at Pro-254.

Drug-resistant variants of thymidylate synthase (TS) can potentially be used in gene therapy applications to decrease the myelosuppressive side effects of TS-directed anticancer agents or to select genetically modified cells in vivo. Mutations of proline 303 of human TS confer resistance to TS-directed fluoropyrimidines and antifolates (). We generated the corresponding variants in Escherichia coli TS (ecTS), position 254, to better understand the mechanism by which mutations at this residue confer resistance. In addition, because ecTS is intrinsically resistant to several antifolates when compared with human TS, we suspected that greater resistance could be achieved with the bacterial enzyme. The P254L enzyme conferred >100-fold resistance to both raltitrexed and 5-fluoro-2'-deoxyuridine (FdUrd) compared with wild-type ecTS. Four additional mutants (P254F, P254S, P254G, and P254D), each of which complemented growth of a TS-deficient cell line, were generated, isolated, and characterized. Steady-state values of K(m) for dUMP and k(cat) were not substantially different among the variants and were comparable with the wild-type values, but K(m) for methylenetetrahydrofolate (CH(2)H(4)PteGlu) was >10-fold higher for P254D. Values of k(on) and k(off) for nucleotide binding, which were obtained by stopped-flow spectroscopy, were virtually unchanged among the mutants. Drastic differences were observed for CH(2)H(4)PteGlu binding, with K(d) values >15-fold higher than observed with the wild-type enzyme; surprisingly, the proposed isomerization reaction that is very evident for the wild-type enzyme is not observed with P254S. The decrease in affinity for CH(2)H(4)PteGlu correlates well with K(i) values obtained for three TS-directed inhibitors. These results show that mutations at Pro-254 specifically affect the initial binding interactions between enzyme and cofactor and also alter the ability of the mutant enzymes to undergo conformational changes that occur on ternary complex formation. The crystal structure of P254S was determined at 1.5 A resolution and is the most precise structure of TS available. When compared with wild-type TS, the structure shows local conformational changes affecting mostly Asp-253; its carbonyl is rotated approximately 40 degrees, and the side chain forms an ion pair with Arg-225.

Crystal structure of human prostatic acid phosphatase .

BACKGROUND: Prostatic acid phosphatase (hPAP) is a major product of the human prostate gland, yet its physiological substrate remains unknown. METHODS: Human PAP, purified from semen, was crystallized using polyethylene glycol as the precipitant and its crystal structure was determined using X-ray diffraction. The structure was refined at 3.1 A resolution to R = 16% and R(free) = 27%. RESULTS: The structure of hPAP is similar to that of other known histidine phosphatases, and the positions of its catalytic residues are conserved. N-linked carbohydrates are present at each of the possible glycosylation sites. It appears that high-mannose chains are attached to Asn 62 and Asp 301, while complex chains are at Asn 188. CONCLUSIONS: The similarity of the three-dimensional structures of rat PAP and human PAP indicates that the mechanistic analyses of the catalytic mechanism proposed for the rat enzyme should be extended to the human enzyme without reservations. The crystallographic data allowed the correlation of attachment sites of N-linked carbohydrate chains with a given carbohydrate type. The carbohydrates of the protein produced in the prostate cells and in the baculovirus expression system appear to differ at the site of complex carbohydrates attachment.

Structural origins of L(+)-tartrate inhibition of human prostatic acid phosphatase.

Acid phosphatase activity in the blood serum is usually separated into tartrate-resistant and tartrate-refractory, which is reported as the prostatic acid phosphatase level. Human prostatic acid phosphatase crystals soaked in N-propyl-L-tartramate were used to collect x-ray diffraction data to 2.9 A resolution under cryogenic conditions. Positive difference electron density, corresponding to the inhibitor, was found. The quality of the electron density maps clearly shows the orientation of the carboxylate and N-propyl-substituted amide groups. The hydroxyl group attached to C3 forms two crucial hydrogen bonds with Arg-79 and His-257. Previous crystallographic studies compiled on the tartrate-rat prostatic acid phosphatase binary complex (Lindqvist, Y., Schneider, G., and Vihko, P. (1993) J. Biol. Chem. 268, 20744-20746) erroneously positioned D-tartrate into the active site. Modeling studies have shown that the C3 hydroxyl group on the D(-)-stereoisomer of tartrate, which does not significantly inhibit prostatic acid phosphatase, does not form strong hydrogen bonds with Arg-79 or His-257. The structure of human prostatic acid phosphatase, noncovalently bound in N-propyl-L-tartramate, is used to develop inhibitors with higher specificity and potency than L(+)-tartrate.

Molecular characterization of a novel short-chain dehydrogenase/reductase that reduces all-trans-retinal.

The reduction of all-trans-retinal in photoreceptor outer segments is the first step in the regeneration of bleached visual pigments. We report here the cloning of a dehydrogenase, retSDR1, that belongs to the short-chain dehydrogenase/reductase superfamily and localizes predominantly in cone photoreceptors. retSDR1 expressed in insect cells displayed substrate specificities of the photoreceptor all-trans-retinol dehydrogenase. Homology modeling of retSDR1 using the carbonyl reductase structure as a scaffold predicted a classical Rossmann fold for the nucleotide binding, and an N-terminal extension that could facilitate binding of the enzyme to the cell membranes. The presence of retSDR1 in a subset of inner retinal neurons and in other tissues suggests that the enzyme may also be involved in retinol metabolism outside of photoreceptors.

Significance of the enzymatic properties of yeast S39A enolase to the catalytic mechanism.

The S39A mutant of yeast enolase (isozyme 1), prepared by site-directed mutagenesis, has a relative Vmax of 0.01% and an activation constant for Mg2+ ca. 10-fold higher, compared with native enzyme. It is correctly folded. There is little effect of solvent viscosity on activity. We think that the loop Ser36-His43 fails to move to the 'closed' position upon catalytic Mg2+ binding, weakening several electrostatic interactions involved in the mechanism.

Mechanism of enolase: the crystal structure of asymmetric dimer enolase-2-phospho-D-glycerate/enolase-phosphoenolpyruvate at 2.0 A resolution.

Enolase, a glycolytic enzyme that catalyzes the dehydration of 2-phospho-d-glycerate (PGA) to form phosphoenolpyruvate (PEP), is a homodimer in all eukaryotes and many prokaryotes. Here, we report the crystal structure of a complex between yeast enolase and an equilibrium mixture of PGA and PEP. The structure has been refined using 29 854 reflections with an F/sigma(F) of >/=3 to an R of 0.137 with average deviations of bond lengths and bond angles from ideal values of 0.013 A and 3.1 degrees , respectively. In this structure, the dimer constitutes the crystallographic asymmetric unit. The two subunits are similar, and their superposition gives a rms distance between Calpha atoms of 0.91 A. The exceptions to this are the catalytic loop Val153-Phe169 where the atomic positions in the two subunits differ by up to 4 A and the loop Ser250-Gln277, which follows the catalytic loop Val153-Phe169. In the first subunit, the imidazole side chain of His159 is in contact with the phosphate group of the substrate/product molecule; in the other it is separated by water molecules. A series of hydrogen bonds leading to a neighboring enolase dimer can be identified as being responsible for ordering and stabilization of the conformationally different subunits in the crystal lattice. The electron density present in the active site suggests that in the active site with the direct ligand-His159 hydrogen bond PGA is predominantly bound while in the active site where water molecules separate His159 from the ligand the binding of PEP dominates. The structure indicates that the water molecule hydrating carbon-3 of PEP in the PEP --> PGA reaction is activated by the carboxylates of Glu168 and Glu211. The crystals are unique because they have resolved two intermediates on the opposite sides of the transition state.

Effect of site-directed mutagenesis of His373 of yeast enolase on some of its physical and enzymatic properties.

The X-ray structure of yeast enolase shows His373 interacting with a water molecule also held by residues Glu168 and Glu211. The water molecule is suggested to participate in the catalytic mechanism (Lebioda, L. and Stec, B. (1991) Biochemistry 30, 2817-2822). Replacement of His373 with asparagine (H373N enolase) or phenylalanine (H373F enolase) reduces enzymatic activity to ca. 10% and 0.0003% of the native enzyme activity, respectively. H373N enolase exhibits a reduced Km for the substrate, 2-phosphoglycerate, and produces the same absorbance changes in the chromophoric substrate analogues TSP1 and AEP1, relative to native enolase. H373F enolase binds AEP less strongly, producing a smaller absorbance change than native enolase, and reacts very little with TSP. H373F enolase dissociates to monomers in the absence of substrate; H373N enolase subunit dissociation is less than H373F enolase but more than native enolase. Substrate and Mg2+ increase subunit association in both mutants. Differential scanning calorimetric experiments indicate that the interaction with substrate that stabilizes enolase to thermal denaturation involves His373. We suggest that the function of His373 in the enolase reaction may involve hydrogen bonding rather than acid/base catalysis, through interaction with the Glu168/Glu211/H2O system, which produces removal or addition of hydroxyl at carbon-3 of the substrate.

Ion binding induces closed conformation in Pseudomonas 7A glutaminase-asparaginase (PGA): crystal structure of the PGA-SO4(2-)-NH4+ complex at 1.7 A resolution.

Pseudomonas 7A glutaminase-asparaginase (PGA) catalyzes the hydrolysis of D- and L-isomers of glutamine and asparagine. X-ray quality type-1 crystals of PGA have been obtained from 2.0 M ammonium sulfate. The space group is C222(1) with unit-cell dimensions a = 78.62, b = 135.80, and c = 137.88 A. The tetrameric molecule is located on a crystallographic 2-fold axis, and two subunits form the asymmetric portion of the unit cell. The structure was solved by the molecular replacement method and refined at 1.7 A resolution to an R = 19.9% with a good geometry of the model, G = 0.05. The resultant electron density maps enabled us to resolve individual constituent atoms of most residues and introduce minor revisions to the amino acid sequence. The catalytic loop, Thr20-Gly40, is in the closed conformation with excellent electron density in both subunits. A sulfate ion and an ammonium ion are bound in the substrate binding site and interect with the loop. This interaction appears to be responsible for the observed closed conformation. New arguments supporting Thr20 as the catalytic nucleophile in the asparaginase activity are proposed.

Prostatic acid phosphatase: structural aspects of inhibition by L-(+)-tartrate ions.

The crystal structure of the complex between rat-prostatic acid phosphatase (PAP) and L-(+)-tartrate (Lindqvist et al., J. Biol. Chem., 1993, 268, 20744-20746) contains the model of the ligand with incorrect chirality. We report here the correct model and discuss the relation between this model and the model of the inhibitory complexes between PAP and oxy-anions.