Crystallization and preliminary X-ray analysis of D-2-hydroxyacid dehydrogenase from Haloferax mediterranei.

D-2-hydroxyacid dehydrogenase (D2-HDH) from Haloferax mediterranei has been overexpressed in Escherichia coli, solubilized in 8 M urea and refolded by rapid dilution. The protein was purified and crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate or PEG 3350 as precipitant. Two crystal forms representing the free enzyme and the nonproductive ternary complex with alpha-ketohexanoic acid and NAD(+) grew under these conditions. Crystals of form I diffracted to beyond 3.0 A resolution and belonged to the monoclinic space group P2(1), with unit-cell parameters a = 66.0, b = 119.6, c = 86.2 A, beta = 96.3 degrees . Crystals of form II diffracted to beyond 2.0 A resolution and belonged to the triclinic space group P1, with unit-cell parameters a = 66.5, b = 75.2, c = 77.6 A, alpha = 109.1, beta = 107.5, gamma = 95.9 degrees. The calculated values for V(M) and analysis of the self-rotation and self-Patterson functions suggest that the asymmetric unit in both crystal forms contains two dimers related by pseudo-translational symmetry.

Crystallization of the Bacillus subtilis SPP1 bacteriophage helicase loader protein G39P.

The essential helicase loader protein G39P encoded by Bacillus subtilis SPP1 phage has been overproduced in Escherichia coli and purified. The wild-type protein has been crystallized by the hanging-drop vapour-diffusion method in a primitive hexagonal space group, probably P6(1)22/P6(5)22, but the crystals diffract to only 3.4 A and are poorly reproducible. Mass-spectrometric analysis has revealed marked proteolytic cleavage from the C-terminus and the presence of a major species corresponding to deletion of the 14 C-terminal residues. Thus, a new variant of the protein (G39P112) has been engineered that corresponds to a 14-residue C-terminal truncation. The G39P112 variant has also been crystallized but now in a primitive orthorhombic form, probably P2(1)2(1)2 or P2(1)2(1)2(1), with unit-cell parameters a = 85.6, b = 89.7, c = 47.6 A, with diffraction to 2.4 A on a synchrotron source and with greatly improved reproducibility. Calculation of V(M) values for this G39P112 variant suggests the presence of three monomers in the asymmetric unit, corresponding to a solvent content of about 47%. A selenomethionine-incorporated form of the protein has been produced and a full three-wavelength MAD data collection undertaken.

Crystallization and preliminary X-ray analysis of the ytxM gene product from Bacillus subtilis.

The ytxM gene product from Bacillus subtilis has been cloned, expressed in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method using PEG 3350 as the precipitant. Multiple-wavelength anomalous dispersive X-ray data have been collected to 2.0 A resolution on a single selenomethionine-incorporated crystal. This crystal belongs to the primitive orthorhombic system, with approximate unit-cell parameters a = 44.3, b = 90.9, c = 136.1 A, alpha = beta = gamma = 90 degrees and two monomers in the asymmetric unit.

Crystallization and preliminary X-ray analysis of glucose dehydrogenase from Haloferax mediterranei.

Glucose dehydrogenase (E.C. 1.1.1.47; GlcDH) from Haloferax mediterranei has been overexpressed in Escherichia coli, solubilized by the addition of 8 M urea and refolded by rapid dilution. The protein has been purified by conventional techniques and crystallized by the hanging-drop vapour-diffusion method using sodium citrate as the precipitant. Two crystal forms representing the free enzyme and the binary complex with NADP(+) grow under these conditions. Crystals of form I diffract to beyond 3.5 A resolution and belong to the hexagonal space group P622, with unit-cell parameters a = b = 89.1, c = 214.6 A, alpha = beta = 90, gamma = 120 degrees. Crystals of form II diffract to greater than 2.0 A and belong to the orthorhombic space group I222 or I2(1)2(1)2(1), with unit-cell parameters a = 61.8, b = 110.9, c = 151.7 A, alpha = beta = gamma = 90 degrees. Calculated values for V(M) and consideration of the packing for both crystal forms suggests that the asymmetric units in both crystal forms contain a monomer.

The 1.2 A structure of a novel quorum-sensing protein, Bacillus subtilis LuxS.

In bacteria, the regulation of gene expression in response to changes in cell density is called quorum sensing. The autoinducer-2 production protein LuxS, is involved in a novel quorum-sensing system and is thought to catalyse the degradation of S-ribosylhomocysteine to homocysteine and the autoinducer molecule 4,5-dihydroxy-2,3-pentadione. The crystal structure of Bacillus subtilis LuxS has been determined at 1.2 A resolution, together with the binary complexes of LuxS with S-ribosylhomocysteine and homocysteine to 2.2 and 2.3 A resolution, respectively. These structures show that LuxS is a homodimer with an apparently novel fold based on an eight-stranded beta-barrel, flanked by six alpha-helices. Each active site contains a zinc ion coordinated by the conserved residues His54, His58 and Cys126, and includes residues from both subunits. S-ribosylhomocysteine binds in a deep pocket with the ribose moiety adjacent to the enzyme-bound zinc ion. Access to the active site appears to be restricted and possibly requires conformational changes in the protein involving the movement of residues 125-129 and those at the N terminus. The structure contains an oxidised cysteine residue in the active site whose role in the biological process of LuxS has not been determined. The autoinducer-2 signalling pathway has been linked to aspects of bacterial virulence and pathogenicity. The structural data on LuxS will provide opportunities for targeting this enzyme for the rational design of new antibiotics.

The structure and domain organization of Escherichia coli isocitrate lyase.

Enzymes of the glyoxylate-bypass pathway are potential targets for the control of many human diseases caused by such pathogens as Mycobacteria and Leishmania. Isocitrate lyase catalyses the first committed step in this pathway and the structure of this tetrameric enzyme from Escherichia coli has been determined at 2.1 A resolution. E. coli isocitrate lyase, like the enzyme from other prokaryotes, is located in the cytoplasm, whereas in plants, protozoa, algae and fungi this enzyme is found localized in glyoxysomes. Comparison of the structure of the prokaryotic isocitrate lyase with that from the eukaryote Aspergillus nidulans reveals a different domain structure following the deletion of approximately 100 residues from the larger eukaryotic enzyme. Despite this, the active sites of the prokaryotic and eukaryotic enzymes are very closely related, including the apparent disorder of two equivalent segments of the protein that are known to be involved in a conformational change as part of the enzyme's catalytic cycle.

Cloning, purification, crystallization and preliminary crystallographic analysis of Bacillus subtilis LuxS.

LuxS of Bacillus subtilis is a member of a novel family of proteins with a potential role in quorum sensing, controlling important aspects of cellular physiology in a range of microbial species. B. subtilis luxS was cloned, expressed in Escherichia coli, purified and crystallized using the hanging-drop method of vapour diffusion with ammonium sulfate as the precipitant. The crystals belong to one of the enantiomorphic space groups P6(1)22 or P6(5)22, with approximate unit-cell parameters a = b = 63.6, c = 151.5 A and one subunit in the asymmetric unit, corresponding to a packing density of 2.5 A(3) Da(-1). The crystals diffract X-rays to at least 1.55 A resolution on a synchrotron-radiation source. Determination of the structure will provide insights into the key determinants of function of this class of proteins, for which no structures are currently available.

The preparation and crystallization of Fab fragments of a family of mouse esterolytic catalytic antibodies and their complexes with a transition-state analogue.

The Fab fragments of a family of mouse esterolytic monoclonal antibodies MS6-12, MS6-126 and MS6-164 have been obtained by digestion of whole antibodies with papain, purified and crystallized in a range of different forms either alone or in complex with a transition-state analogue. The crystals diffract X-rays to resolutions between 2.1 and 1.2 A and are suitable for structural studies. The determination of these structures could be important in understanding the different catalytic power of each of these related catalytic antibodies.

Analysis of the structure, substrate specificity, and mechanism of squash glycerol-3-phosphate (1)-acyltransferase.

BACKGROUND: Glycerol-3-phosphate (1)-acyltransferase(G3PAT) catalyzes the incorporation of an acyl group from either acyl-acyl carrier proteins (acylACPs) or acyl-CoAs into the sn-1 position of glycerol 3-phosphate to yield 1-acylglycerol-3-phosphate. G3PATs can either be selective, preferentially using the unsaturated fatty acid, oleate (C18:1), as the acyl donor, or nonselective, using either oleate or the saturated fatty acid, palmitate (C16:0), at comparable rates. The differential substrate specificity for saturated versus unsaturated fatty acids seen within this enzyme family has been implicated in the sensitivity of plants to chilling temperatures. RESULTS: The three-dimensional structure of recombinant G3PAT from squash chloroplast has been determined to 1.9 A resolution by X-ray crystallography using the technique of multiple isomorphous replacement and provides the first representative structure of an enzyme of this class. CONCLUSIONS: The tertiary structure of G3PAT comprises two domains, the larger of which, domain II, features an extensive cleft lined by hydrophobic residues and contains at one end a cluster of positively charged residues flanked by a H(X)(4)D motif, which is conserved amongst many glycerolipid acyltransferases. We predict that these hydrophobic and positively charged residues represent the binding sites for the fatty acyl substrate and the phosphate moiety of the glycerol 3-phosphate, respectively, and that the H(X)(4)D motif is a critical component of the enzyme's catalytic machinery.

Crystallization and quaternary structure analysis of an Lrp-like regulatory protein from the hyperthermophile Pyrococcus furiosus.

The LrpA transcriptional regulator from Pyrococcus furiosus, a member of the leucine-responsive regulatory protein (Lrp) family, has been crystallized by the hanging-drop method of vapour diffusion using ammonium sulfate as the precipitant. The crystals belong to the tetragonal system and are in space group I4(1)22, with unit-cell parameters a = b = 104.5, c = 245.1 A. Consideration of the values of V(M) and possible packing of the molecules within the cell suggest that the asymmetric unit contains a dimer. Examination of the behaviour of the protein on gel-filtration columns and analysis of the self-rotation function suggests that the molecule is an octamer in solution at around pH 5. Determination of the structure of this protein will provide insights into the mechanisms responsible for DNA-protein recognition at high temperature and into how the regulatory properties of the Lrp family are modified by the presence or absence of effector molecules.

Interaction of cytochrome bd with carbon monoxide at low and room temperatures: evidence that only a small fraction of heme b595 reacts with CO.

Azotobacter vinelandii is an obligately aerobic bacterium in which aerotolerant dinitrogen fixation requires cytochrome bd. This oxidase comprises two polypeptide subunits and three hemes, but no copper, and has been studied extensively. However, there remain apparently conflicting reports on the reactivity of the high spin heme b(595) with ligands. Using purified cytochrome bd, we show that absorption changes induced by CO photodissociation from the fully reduced cytochrome bd at low temperatures demonstrate binding of the ligand with heme b(595). However, the magnitude of these changes corresponds to the reaction with CO of only about 5% of the heme. CO binding with a minor fraction of heme b(595) is also revealed at room temperature by time-resolved studies of CO recombination. The data resolve the apparent discrepancies between conclusions drawn from room and low temperature spectroscopic studies of the CO reaction with cytochrome bd. The results are consistent with the proposal that hemes b(595) and d form a diheme oxygen-reducing center with a binding capacity for a single exogenous ligand molecule that partitions between the hemes d and b(595) in accordance with their intrinsic affinities for the ligand. In this model, the affinity of heme b(595) for CO is about 20-fold lower than that of heme d.

Crystal structure of the Lrp-like transcriptional regulator from the archaeon Pyrococcus furiosus.

The LrpA protein from the hyperthermophilic archaeon Pyrococcus furiosus belongs to the Lrp/AsnC family of transcriptional regulatory proteins, of which the Escherichia coli leucine-responsive regulatory protein is the archetype. Its crystal structure has been determined at 2.9 A resolution and is the first for a member of the Lrp/AsnC family, as well as one of the first for a transcriptional regulator from a hyperthermophile. The structure consists of an N-terminal domain containing a helix-turn-helix (HtH) DNA-binding motif, and a C-terminal domain of mixed alpha/beta character reminiscent of a number of RNA- and DNA-binding domains. Pyrococcus furiosus LrpA forms a homodimer mainly through interactions between the antiparallel beta-sheets of the C-terminal domain, and further interactions lead to octamer formation. The LrpA structure suggests how the protein might bind and possibly distort its DNA substrate through use of its HtH motifs and control gene expression. A possible location for an effector binding site is proposed by using sequence comparisons with other members of the family coupled to mutational analysis.

Crystallization of the dI component of transhydrogenase, a proton-translocating membrane protein.

Nicotinamide nucleotide transhydrogenase couples the exchange of a hydride-ion equivalent between NAD(H) and NADP(H) to the translocation of protons across an energy-transducing membrane. Peripheral components of 380 and 200 residues bind NAD(H) (dI) and NADP(H) (dIII), respectively, while a third component forms a membrane-spanning region (dII). The NAD(H)-binding component dI of Rhodospirillum rubrum transhydrogenase has been crystallized in a form which diffracts to beyond 3.0 A resolution and is in space group P2 or P2(1), with unit-cell parameters a = 69.3, b = 117.8, c = 106.6 A, beta = 107.2 degrees and two dimers in the asymmetric unit. The sequence of the dI component is similar to that of alanine dehydrogenase. A full structure determination will lead to important information on the mode of action of this proton pump and will permit the comparison of the structure-function relationships of dI with those of alanine dehydrogenase.

The crystal structure and active site location of isocitrate lyase from the fungus Aspergillus nidulans.

BACKGROUND: Isocitrate lyase catalyses the first committed step of the carbon-conserving glyoxylate bypass, the Mg(2+)-dependent reversible cleavage of isocitrate into succinate and glyoxylate. This metabolic pathway is an inviting target for the control of a number of diseases, because the enzymes involved in this cycle have been identified in many pathogens including Mycobacterium leprae and Leishmania. RESULTS: As part of a programme of rational drug design the structure of the tetrameric Aspergillus nidulans isocitrate lyase and its complex with glyoxylate and a divalent cation have been solved to 2.8 A resolution using X-ray diffraction. Each subunit comprises two domains, one of which adopts a folding pattern highly reminiscent of the triose phosphate isomerase (TIM) barrel. A 'knot' between subunits observed in the three-dimensional structure, involving residues towards the C terminus, implies that tetramer assembly involves considerable flexibility in this part of the protein. CONCLUSIONS: Difference Fourier analysis together with the pattern of sequence conservation has led to the identification of both the glyoxylate and metal binding sites and implicates the C-terminal end of the TIM barrel as the active site, which is consistent with studies of other enzymes with this fold. Two disordered regions of the polypeptide chain lie close to the active site, one of which includes a critical cysteine residue suggesting that conformational rearrangements are essential for catalysis. Structural similarities between isocitrate lyase and both PEP mutase and enzymes belonging to the enolase superfamily suggest possible relationships in aspects of the mechanism.

Crystallization of the NADP-dependent beta-keto acyl-carrier protein reductase from Brassica napus.

The NADP-dependent beta-keto acyl-carrier protein reductase (BKR) from Brassica napus has been crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol of average molecular weight 1500 as the precipitant. The crystals belong to the hexagonal space group P6(4)22, with unit-cell parameters a = b = 129. 9, c = 93.1 A, alpha = beta = 90, gamma = 120 degrees. Calculated values for V(m), the use of rotation and translation functions and consideration of the packing suggest that the asymmetric unit contains a monomer. The crystals diffract to beyond 2.8 A resolution and are more amenable to X-ray diffraction analysis than those reported previously for the Escherichia coli enzyme. The structure determination of B. napus BKR will provide important insights into the catalytic mechanism of the enzyme and into the evolution of the fatty-acid elongation cycle by comparisons with the other oxidoreductase of the pathway, enoyl acyl-carrier protein reductase (ENR).

Mutagenesis of squash (Cucurbita moschata) glycerol-3-phosphate acyltransferase (GPAT) to produce an enzyme with altered substrate selectivity.

In an attempt to rationalize the relationship between structure and substrate selectivity of glycerol-3-phosphate acyltransferase (GPAT, 1AT, EC 2.3.1.15) we have cloned a number of cDNAs into the pET overexpression system using a PCR-based approach. Following assay of the recombinant enzyme we noted that the substrate selectivity of the squash (Cucurbita moschata) enzyme had altered dramatically. This form of GPAT has now been crystallized and its full three-dimensional structure elucidated. Since we now have two forms of the enzyme that display different substrate selectivities this should provide a powerful tool to determine the basis of the selectivity changes. Kinetic and structural analyses are currently being performed to rationalize the changes which have taken place.

Structure determination of the glutamate dehydrogenase from the hyperthermophile Thermococcus litoralis and its comparison with that from Pyrococcus furiosus.

Glutamate dehydrogenase catalyses the oxidative deamination of glutamate to 2-oxoglutarate with concomitant reduction of NAD(P)(+), and has been shown to be widely distributed in nature across species ranging from psychrophiles to hyperthermophiles. Extensive characterisation of this enzyme isolated from hyperthermophilic organisms has led to its adoption as a model system for analysing the determinants of thermal stability. The crystal structure of the extremely thermostable glutamate dehydrogenase from Thermococcus litoralis has been determined at 2.5 A resolution, and has been compared to that from the hyperthermophile Pyrococcus furiosus. The two enzymes are 87 % identical in sequence, yet differ 16-fold in their half-lives at 104 degrees C. This is the first reported comparative analysis of the structures of a multisubunit enzyme from two closely related yet distinct hyperthermophilies. The less stable T. litoralis enzyme has a decreased number of ion pair interactions; modified patterns of hydrogen bonding resulting from isosteric sequence changes; substitutions that decrease packing efficiency; and substitutions which give rise to subtle but distinct shifts in both main-chain and side-chain elements of the structure. This analysis provides a rational basis to test ideas on the factors that confer thermal stability in proteins through a combination of mutagenesis, calorimetry, and structural studies.

Crystallization of Escherichia coli RuvA complexed with a synthetic Holliday junction.

During homologous recombination in Escherichia coli the RuvA, B and C proteins interact specifically with the Holliday junction formed by the action of RecA to promote the strand-exchange reaction. RuvA, a homotetrameric protein of molecular weight 88 kDa, has been overexpressed in E. coli, purified and co-crystallized with a synthetic Holliday junction substrate made from four 18-base deoxyoligonucleotides. Crystals were grown using the hanging-drop vapour-diffusion method with sodium acetate as the precipitant. The crystals diffract to a resolution of 6 A and belong to the monoclinic system, space group C2, with cell parameters a = 148, b = 148, c = 106 A and beta = 123 degrees. The X-ray analysis of these crystals should reveal the structure of the Holliday junction and its mode of binding to RuvA, providing new insights into the molecular mechanism of genetic recombination.

Analysis of the structure and substrate binding of Phormidium lapideum alanine dehydrogenase.

The structure of the hexameric L-alanine dehydrogenase from Phormidium lapideum reveals that the subunit is constructed from two domains, each having the common dinucleotide binding fold. Despite there being no sequence similarity, the fold of alanine dehydrogenase is closely related to that of the family of D-2-hydroxyacid dehydrogenases, with a similar location of the active site, suggesting that these enzymes are related by divergent evolution. L-alanine dehydrogenase and the 2-hydroxyacid dehydrogenases also use equivalent functional groups to promote substrate recognition and catalysis. However, they are arranged differently on the enzyme surface, which has the effect of directing opposite faces of the keto acid to the dinucleotide in each case, forcing a change in absolute configuration of the product.