The 2.8 A crystal structure of peroxisomal 3-ketoacyl-CoA thiolase of Saccharomyces cerevisiae: a five-layered alpha beta alpha beta alpha structure constructed from two core domains of identical topology.

BACKGROUND: The peroxisomal enzyme 3-ketoacyl-coenzyme A thiolase of the yeast Saccharomyces cerevisiae is a homodimer with 417 residues per subunit. It is synthesized in the cytosol and subsequently imported into the peroxisome where it catalyzes the last step of the beta-oxidation pathway. We have determined the structure of this thiolase in order to study the reaction mechanism, quaternary associations and intracellular targeting of thiolases generally, and to understand the structural basis of genetic disorders associated with human thiolases. RESULTS: Here we report the crystal structure of unliganded yeast thiolase refined at 2.8 A resolution. The enzyme comprises three domains; two compact core domains having the same fold and a loop domain. Each of the two core domains is folded into a mixed five-stranded beta-sheet covered on each side by helices and the two are assembled into a five-layered alpha beta alpha beta alpha structure. The central layer is formed by two helices, which point with their amino termini towards the active site. The loop domain, which is to some extent stabilized by interactions with the other subunit, runs over the surface of the two core domains, encircling the active site of its own subunit. CONCLUSIONS: The crystal structure of thiolase shows that the active site is a shallow pocket, shaped by highly conserved residues. Two conserved cysteines and a histidine at the floor of this pocket probably play key roles in the reaction mechanism. The two active sites are on the same face of the dimer, far from the amino and carboxyl termini of both subunits and the disordered amino-terminal import signal sequence.

The crystal structure of an engineered monomeric triosephosphate isomerase, monoTIM: the correct modelling of an eight-residue loop.

BACKGROUND: The triosephosphate isomerase (TIM) fold is found in several different classes of enzymes, most of which are oligomers; TIM itself always functions as a very tight dimer. It has recently been shown that a monomeric form of TIM ('monoTIM') can be constructed by replacing a 15-residue interface loop, loop-3, with an eight-residue fragment; modelling suggests that this should result in a short strain-free turn, resulting in the subsequent helix, helix-A3, having an additional turn at its amino terminus. RESULTS: The crystal structure of monoTIM shows that it retains the characteristic TIM-barrel (betaalpha)8-fold and that the new loop has a structure very close to that predicted. Two other interface loops, loop-1 and loop-4, which contain the active site residues Lys13 and His95, respectively, show significant changes in structure in monoTIM compared with dimeric wild-type TIM. CONCLUSION: The observed structural differences between monoTIM and wild-type TIM indicate that the dimeric appearance of TIM determines the location and conformation of two of the four catalytic residues.

Three new crystal structures of point mutation variants of monoTIM: conformational flexibility of loop-1, loop-4 and loop-8.

BACKGROUND: Wild-type triosephosphate isomerase (TIM) is a very stable dimeric enzyme. This dimer can be converted into a stable monomeric protein (monoTIM) by replacing the 15-residue interface loop (loop-3) by a shorter, 8-residue, loop. The crystal structure of monoTIM shows that two active-site loops (loop-1 and loop-4), which are at the dimer interface in wild-type TIM, have acquired rather different structural properties. Nevertheless, monoTIM has residual catalytic activity. RESULTS: Three new structures of variants of monoTIM are presented, a double-point mutant crystallized in the presence and absence of bound inhibitor, and a single-point mutant in the presence of a different inhibitor. These new structures show large structural variability for the active-site loops, loop-1, loop-4 and loop-8. In the structures with inhibitor bound, the catalytic lysine (Lys13 in loop-1) and the catalytic histidine (His95 in loop-4) adopt conformations similar to those observed in wild-type TIM, but very different from the monoTIM structure. CONCLUSIONS: The residual catalytic activity of monoTIM can now be rationalized. In the presence of substrate analogues the active-site loops, loop-1, loop-4 and loop-8, as well as the catalytic residues, adopt conformations similar to those seen in the wild-type protein. These loops lack conformational flexibility in wild-type TIM. The data suggest that the rigidity of these loops in wild-type TIM, resulting from subunit-subunit contacts at the dimer interface, is important for optimal catalysis.

Crystallographic studies of 3-ketoacylCoA thiolase from yeast Saccharomyces cerevisiae.

Good diffracting crystals of 3-ketoacylCoA thiolase (EC 2.3.1.16) from yeast Saccharomyces cerevisiae have been obtained. The crystals diffract to at least 2.4 A. The space group of these crystals is P2(1)2(1)2(1), with cell dimensions a = 71.8 A, b = 93.8 A and c = 119.9 A. There is one dimer per asymmetric unit.

Crystallization and preliminary X-ray diffraction studies of mitochondrial short-chain delta 3,delta 2-enoyl-CoA isomerase from rat liver.

Crystals of short-chain delta 3,delta 2-enoyl-CoA isomerase (EC 5.3.3.8) from rat liver mitochondria have been grown using the hanging-drop vapour diffusion technique. The enoyl-CoA isomerase is an auxiliary enzyme in the beta-oxidation pathway of fatty acid metabolism, and catalyzes the isomerization of unsaturated fatty acids to produce the metabolizable delta 2-trans isomer. The crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions a = 47.9, b = 118.4 and c = 164.8 A, and diffract to 3 A.

The 1.8 A crystal structure of the dimeric peroxisomal 3-ketoacyl-CoA thiolase of Saccharomyces cerevisiae: implications for substrate binding and reaction mechanism.

The dimeric, peroxisomal 3-ketoacyl-CoA thiolase catalyses the conversion of 3-ketoacyl-CoA into acyl-CoA, which is shorter by two carbon atoms. This reaction is the last step of the beta-oxidation pathway. The crystal structure of unliganded peroxisomal thiolase of the yeast Saccharomyces cerevisiae has been refined at 1.8 A resolution. An unusual feature of this structure is the presence of two helices, completely buried in the dimer and sandwiched between two beta-sheets. The analysis of the structure shows that the sequences of these helices are not hydrophobic, but generate two amphipathic helices. The helix in the N-terminal domain exposes the polar side-chains to a cavity at the dimer interface, filled with structured water molecules. The central helix in the C-terminal domain exposes its polar residues to an interior polar pocket. The refined structure has also been used to predict the mode of binding of the substrate molecule acetoacetyl-CoA, as well as the reaction mechanism. From previous studies it is known that Cys125, His375 and Cys403 are important catalytic residues. In the proposed model the acetoacetyl group fits near the two catalytic cysteine residues, such that the oxygen atoms point towards the protein interior. The distance between SG(Cys125) and C3(acetoacetyl-CoA) is 3.7 A. The O2 atom of the docked acetoacetyl group makes a hydrogen bond to N(Gly405), which would favour the formation of the covalent bond between SG(Cys125) and C3(acetoacetyl-CoA) of the intermediate complex of the two-step reaction. The CoA moiety is proposed to bind in a groove on the surface of the protein molecule. Most of the interactions of the CoA molecule are with atoms of the loop domain. The three phosphate groups of the CoA moiety are predicted to interact with side-chains of lysine and arginine residues, which are conserved in the dimeric thiolases.

Comparison of the structures and the crystal contacts of trypanosomal triosephosphate isomerase in four different crystal forms.

Triosephosphate isomerase (TIM) is a dimeric enzyme consisting of 2 identical subunits. Trypanosomal TIM can be crystallized in 4 different spacegroups: P2(1)2(1)2(1), C2(big cell), C2(small cell), and P1. The P1 crystal form only grows in the presence of 1.4 M DMSO; there are 2 DMSO binding sites per subunit. The structures have been refined at a resolution of 1.83 A, 2.10 A, 2.13 A, and 1.80 A, respectively. In the 4 different spacegroups the TIM subunit can be observed in the context of 7 different crystallographic environments. In the C2 cells, the dimer 2-fold axis coincides with a crystallographic 2-fold axis. The similarities and differences of the 7 subunits are discussed. In 6 subunits the flexible loop (loop 6) is open, whereas in the P2(1)2(1)2(1) cell, the flexible loop of subunit 2 is in an almost closed conformation. The crystal contacts in the 4 different crystal forms are predominantly generated by polar residues in loops. A statistical analysis of the residues involved in crystal contacts shows that, in particular, serines are frequently involved in these interactions; 19% of the exposed serines are involved in crystal contacts.

Primary structure of elongation factor 1 gamma from Artemia.

Complementary DNA corresponding to elongation factor 1 gamma, which forms a complex with EF-1 beta, has been cloned. A lambda gt11 cDNA library has been screened with an antiserum against EF-1 beta gamma. The derived amino acid sequence of EF-1 gamma corresponds to 429 amino acids excluding the initiator methionine, which is absent in the mature protein. About half of the protein was sequenced by direct protein sequence analysis. No clear homology with any other protein was found.

The crystal structure of human CskSH3: structural diversity near the RT-Src and n-Src loop.

SH3 domains are modules occurring in diverse proteins, ranging from cytoskeletal proteins to signaling proteins, such as tyrosine kinases. The crystal structure of the SH3 domain of Csk (c-Src specific tyrosine kinase) has been refined at a resolution of 2.5 A, with an R-factor of 22.4%. The structure is very similar to the FynSH3 crystal structure. When comparing CskSH3 and FynSH3 it is seen that the structural and charge differences of the RT-Src loop and the n-Src loop, near the conserved Trp47, correlate with different binding properties of these SH3 domains. The structure comparison suggests that those glycines and acid residues which are very well conserved in the SH3 sequences are important for the stability of the SH3 fold.

An interface point-mutation variant of triosephosphate isomerase is compactly folded and monomeric at low protein concentrations.

Wild-type trypanosomal triosephosphate isomerase (wtTIM) is a very tight dimer. The interface residue His-47 of wtTIM has been mutated into an asparagine. Ultracentrifugation data show that this variant (H47N) only dimerises at protein concentrations above 3 mg/ml. H47N has been characterised at a protein concentration where it is predominantly a monomer. Circular dichroism measurements in the near-UV and far-UV show that this monomer is a compactly folded protein with secondary structure similar as in wtTIM. The thermal stability of the monomeric H47N is decreased compared to wtTIM: temperature gradient gel electrophoresis (TGGE) measurements give Tm-values of 41 degrees C for wtTIM, whereas the Tm-value for the monomeric form of H47N is approximately 7 degrees C lower.

Structures of the "open" and "closed" state of trypanosomal triosephosphate isomerase, as observed in a new crystal form: implications for the reaction mechanism.

The structure of trypanosomal triosephosphate isomerase (TIM) has been solved at a resolution of 2.1A in a new crystal form grown at pH 8.8 from PEG6000. In this new crystal form (space group C2, cell dimensions 94.8 A, 48.3 A, 131.0 A, 90.0 degrees, 100.3 degrees, 90.0 degrees), TIM is present in a ligand-free state. The asymmetric unit consists of two TIM subunits. Each of these subunits is part of a dimer which is sitting on a crystallographic twofold axis, such that the crystal packing is formed from two TIM dimers in two distinct environments. The two constituent monomers of a given dimer are, therefore, crystallographically equivalent. In the ligand-free state of TIM in this crystal form, the two types of dimer are very similar in structure, with the flexible loops in the "open" conformation. For one dimer (termed molecule-1), the flexible loop (loop-6) is involved in crystal contacts. Crystals of this type have been used in soaking experiments with 0.4 M ammonium sulphate (studied at 2.4 A resolution), and with 40 microM phosphoglycolohydroxamate (studied at 2.5 A resolution). It is found that transfer to 0.4 M ammonium sulphate (equal to 80 times the Ki of sulphate for TIM), gives rise to significant sulphate binding at the active site of one dimer (termed molecule-2), and less significant binding at the active site of the other. In neither dimer does sulphate induce a "closed" conformation. In a mother liquor containing 40 microM phosphoglycolohydroxamate (equal to 10 times the Ki of phosphoglycolohydroxamate for TIM), an inhibitor molecule binds at the active site of only that dimer of which the flexible loop is free from crystal contacts (molecule-2). In this dimer, it induces a closed conformation. These three structures are compared and discussed with respect to the mode of binding of ligand in the active site as well as with respect to the conformational changes resulting from ligand binding.

An improved procedure for the X-ray microanalysis of acid phosphatase activity in lysosomes.

The useful detection of acid phosphatase activity with cerium as a capturing agent is confirmed. By introducing a freeze step in combination with a preincubation, reliably localized, lysosomal precipitates are obtained and aspecific ones prevented. Short (t less than 1 h) postfixation with either OsO4 plus K4Fe (CN)6 or OsO4 plus aminotriazole, added to lysosomal cerium localization a high membrane contrast. The detection of cerium by X-ray microanalysis is improved by a better spectral separation of the osmium (M alpha) and cerium (L alpha) peaks.

Crystallization experiments with 2-enoyl-CoA hydratase, using an automated 'fast-screening' crystallization protocol.

A convenient method for screening crystallization conditions using an automated fast-screen protocol has been implemented and tested on an enoyl-CoA hydratase. The crystallization solutions for the initial screening and subsequent optimizations are prepared using a crystallization robot. Enoyl-CoA hydratase (E.C. 4.2.1.17), purified from rat-liver mitochondria, is one of the enzymes from the beta-oxidation pathway of fatty-acid metabolism; it catalyzes the reversible hydration of 2-trans-enoyl-CoA's to L-3-hydroxy-acyl-CoA's. Different crystal forms, diffracting to 3.0 A, were obtained.

Crystal structure of enoyl-coenzyme A (CoA) hydratase at 2.5 angstroms resolution: a spiral fold defines the CoA-binding pocket.

The crystal structure of rat liver mitochondrial enoyl-coenzyme A (CoA) hydratase complexed with the potent inhibitor acetoacetyl-CoA has been refined at 2.5 angstroms resolution. This enzyme catalyses the reversible addition of water to alpha,beta-unsaturated enoyl-CoA thioesters, with nearly diffusion-controlled reaction rates for the best substrates. Enoyl-CoA hydratase is a hexamer of six identical subunits of 161 kDa molecular mass for the complex. The hexamer is a dimer of trimers. The monomer is folded into a right-handed spiral of four turns, followed by two small domains which are involved in trimerization. Each turn of the spiral consists of two beta-strands and an alpha-helix. The mechanism for the hydratase/dehydratase reaction follows a syn-stereochemistry, a preference that is opposite to the nonenzymatic reaction. The active-site architecture agrees with this stereochemistry. It confirms the importance of Glu164 as the catalytic acid for providing the alpha-proton during the hydratase reaction. It also shows the importance of Glu144 as the catalytic base for the activation of a water molecule in the hydratase reaction. The comparison of an unliganded and a liganded active site within the same crystal form shows a water molecule in the unliganded subunit. This water molecule is bound between the two catalytic glutamates and could serve as the activated water during catalysis.

Protein engineering with monomeric triosephosphate isomerase (monoTIM): the modelling and structure verification of a seven-residue loop.

Protein engineering experiments have been carried out with loop-1 of monomeric triosephosphate isomerase (monoTIM). Loop-1 of monoTIM is disordered in every crystal structure of liganded monoTIM, but in the wild-type TIM it is a very rigid dimer interface loop. This loop connects the first beta-strand with the first alpha-helix of the TIM-barrel scaffold. The first residue of this loop, Lys13, is a conserved catalytic residue. The protein design studies with loop-1 were aimed at rigidifying this loop such that the Lys13 side chain points in the same direction as seen in wild type. The modelling suggested that the loop should be made one residue shorter. With the modelling package ICM the optimal sequence of a new seven-residue loop-1 was determined and its structure was predicted. The new variant could be expressed and purified and has been characterized. The catalytic activity and stability are very similar to those of monoTIM. The crystal structure (at 2.6 A resolution) shows that the experimental loop-1 structure agrees well with the modelled loop-1 structure. The direct superposition of the seven loop residues of the modelled and experimental structures results in an r.m.s. difference of 0.5 A for the 28 main chain atoms. The good agreement between the predicted structure and the crystal structure shows that the described modelling protocol can be used successfully for the reliable prediction of loop structures.

Modular mutagenesis of a TIM-barrel enzyme: the crystal structure of a chimeric E. coli TIM having the eighth beta alpha-unit replaced by the equivalent unit of chicken TIM.

The crystal structure of a hybrid Escherichia coli triosephosphate isomerase (TIM) has been determined at 2.8 A resolution. The hybrid TIM (ETIM8CHI) was constructed by replacing the eighth beta alpha-unit of E. coli TIM with the equivalent unit of chicken TIM. This replacement involves 10 sequence changes. One of the changes concerns the mutation of a buried alanine (Ala232 in strand 8) into a phenylalanine. The ETIM8CHI structure shows that the A232F sequence change can be incorporated by a side-chain rotation of Phe224 (in helix 7). No cavities or strained dihedrals are observed in ETIM8CHI in the region near position 232, which is in agreement with the observation that ETIM8CHI and E.coli TIM have similar stabilities. The largest CA (C-alpha atom) movements, approximately 3 A, are seen for the C-terminal end of helix 8 (associated with the outward rotation of Phe224) and for the residues in the loop after helix 1 (associated with sequence changes in helix 8). From the structure it is not clear why the kcat of ETIM8CHI is 10 times lower than in wild type E.coli TIM.

Structure of triosephosphate isomerase from Escherichia coli determined at 2.6 A resolution.

The structure of triosephosphate isomerase (TIM) from the organism Escherichia coli has been determined at a resolution of 2.6 A. The structure was solved by the molecular replacement method, first at 2.8 A resolution with a crystal grown by the technique of hanging-drop crystallization from a mother liquor containing the transition-state analogue 2-phosphoglycolate (2PG). As a search model in the molecular replacement calculations, the refined structure of TIM from Trypanosoma brucei, which has a sequence identity of 46% compared to the enzyme from E. coli, was used. An E. coli TIM crystal grown in the absence of 2PG, diffracting to 2.6 A resolution, was later obtained by application of the technique of macro-seeding using a seed crystal grown from a mother liquor without 2PG. The final 2.6 A model has a crystallographic R factor of 11.9%, and agrees well with standard stereochemical parameters. The structure of E. coli TIM suggests the importance of residues which favour helix initiation for the formation of the TIM fold. In addition, TIM from E. coli shows peculiarities in its dimer interface, and in the packing of core residues within the beta-barrel.

Selective interaction of glycosomal enzymes from Trypanosoma brucei with hydrophobic cyclic hexapeptides.

Hydrophobic cyclic hexapeptides have been reported to selectively inhibit glycosomal triosephosphate isomerase from Trypanosoma brucei (Kuntz et al, 1992, Eur. J. Biochem., 207, 441-447). Here it is shown that this inhibition is not due to a specific interaction between the enzyme and soluble hydrophobic cyclic hexapeptides, but that it is the result of a coprecipitation of trypanosome triosephosphate isomerase with cyclic hexapeptides when the solubilities of the latter are exceeded. A study of the interaction of these hexapeptides with other glycosomal enzymes revealed that several of them, such as phosphoglycerate kinase and hexokinase, also coprecipitated with these peptides, whereas most of the homologous enzymes from other organisms did not coprecipitate, nor were they inactivated.

Overexpression of trypanosomal triosephosphate isomerase in Escherichia coli and characterisation of a dimer-interface mutant.

In this paper, the successful expression of trypanosomal triosephosphate isomerase (TIM) from Trypanosoma brucei brucei to high yield in Escherichia coli, using a T7-polymerase-based expression system, is described. Overexpressed trypanosomal TIM is fully active. The measured physicochemical properties of this recombinant TIM and TIM purified from trypanosomes are indistinguishable. Crystals of recombinant TIM have been grown in the presence of 2.4 M ammonium sulphate under the same conditions as for trypanosomally expressed TIM. The recombinant TIM crystal structure has been refined at 0.23 nm resolution; no differences were detected between this structure and the original crystal structure. A TIM mutant was made in which a unique dimer-interface histidine residue (His47) was changed into an asparagine. This variant ([H47N]TIM) could be expressed and purified to homogeneity by a procedure which was somewhat different from the purification of recombinant wild-type TIM. It is shown that the [H47N]TIM dimer is considerably less stable than wild-type trypanosomal TIM. The catalytic activity of [H47N]TIM is concentration dependent. The dilution-dependent inactivation is reversible. His47 is involved in a water-mediated hydrogen bond with Asp385 of the other subunit. The lower stability of the [H47N]TIM dimer implies that this water-mediated hydrogen bond is important for the stability of the TIM dimer.

Structural and mutagenesis studies of leishmania triosephosphate isomerase: a point mutation can convert a mesophilic enzyme into a superstable enzyme without losing catalytic power.

The dimeric enzyme triosephosphate isomerase (TIM) has a very tight and rigid dimer interface. At this interface a critical hydrogen bond is formed between the main chain oxygen atom of the catalytic residue Lys13 and the completely buried side chain of Gln65 (of the same subunit). The sequence of Leishmania mexicana TIM, closely related to Trypanosoma brucei TIM (68% sequence identity), shows that this highly conserved glutamine has been replaced by a glutamate. Therefore, the 1.8 A crystal structure of leishmania TIM (at pH 5.9) was determined. The comparison with the structure of trypanosomal TIM shows no rearrangements in the vicinity of Glu65, suggesting that its side chain is protonated and is hydrogen bonded to the main chain oxygen of Lys13. Ionization of this glutamic acid side chain causes a pH-dependent decrease in the thermal stability of leishmania TIM. The presence of this glutamate, also in its protonated state, disrupts to some extent the conserved hydrogen bond network, as seen in all other TIMs. Restoration of the hydrogen bonding network by its mutation to glutamine in the E65Q variant of leishmania TIM results in much higher stability; for example, at pH 7, the apparent melting temperature increases by 26 degrees C (57 degrees C for leishmania TIM to 83 degrees C for the E65Q variant). This mutation does not affect the kinetic properties, showing that even point mutations can convert a mesophilic enzyme into a superstable enzyme without losing catalytic power at the mesophilic temperature.