Search

UDP-glucose pyrophosphorylase (UGPase) catalyzes the synthesis of UDP-glucose, an essential metabolite in all living organisms. An X-ray crystallographic study of UGPase from Helicobacter pylori has been performed in order to elucidate its role in the regulation of this important metabolic pathway. UGPase was crystallized from 0.1 M sodium acetate trihydrate pH 4.6, 2.0 M ammonium sulfate and 0.1 M guanidine-HCl. According to diffraction data collected at a resolution of 2.9 Å using a synchrotron-radiation source, the crystal belongs to the orthorhombic space group P212121, with unit-cell parameters a = 91.47, b = 98.61, c = 245.70 Å, α =β = γ = 90.0°. [ABSTRACT FROM AUTHOR]

CIpX, a member of the HSP (heat-shock protein) 100 family, functions as a molecular chaperone and is a regulatory subunit of the CIpXP protease. To understand the chaperone and regulatory mechanisms of CIpX, Helicobacter pylori CIpX has been over-expressed in Escherichia coli and crystallized at 295 K using (NH[SUB4])[SUB2]HPO[SUB4] as precipitant, X-ray diffraction data have been collected to 2.6Å resolution using a synchrotron-radiation source. The crystals belong to the hexagonal space group P6[SUB5] or P6[SUB1], with unit-cell parameters a = b = 78.52(04), c = 131.51(09) Å, α = &beta = 90, γ = 120°. The crystallographic asymmetric unit contains one molecule of CIpX, with a corresponding V[SUBM] of 2.78Å[SUB3] Da[SUP-1] and a solvent content of 55.8%. [ABSTRACT FROM AUTHOR]

HtrA (high-temperature requirement A) is a widely distributed heat- shock protein which has both molecular-chaperone and proteolytic activities. It is composed of two PDZ domains essential for oligomerization and a protease domain. To understand the molecular basis of the dual function of HtrA, the protease domain of T. maritima HtrA has been crystallized. X-ray diffraction data have been collected to 2.7 Å resolution using a synchrotron-radiation source. Crystals belong to the cubic space group P213, with unit-cell parameters a = b = c = 120.55 (8) Å. The asymmetric unit contains two protease domains, with a corresponding VM of 2.80 ų Da-1 and a solvent content of 56.1%. Ribosome recycling factor (RRF) plays a central role during the recycling of ribosomes in the final step of protein biosynthesis in prokaryotes and is therefore a favourable target for the development of new antibiotics. The crystal structure of Escherichia coli RRF has been reported to have an open L-shaped conformation, while other RRFs from thermophilic bacteria have a strict L-shaped conformation [Yun et al. (2000), Acta Cryst. D56, 84-85]. Wild-type E. coli RRF has so far not been crystallized free from bound detergent. Here, a mutant of RRF, Arg132Gly, has been crystallized without any detergent. A complete data set from a crystal of this mutant obtained by the hanging-drop vapour-diffusion method has been collected at 2.2 A resolution using synchrotron radiation at 100 K. The crystal belongs to the monoclinic space group P21, with unit-cell parameters a = 46.02, b = 49.27, c = 49.37 A, β = 110.1°. The currently refined structure indicates that RRF has a tRNA-like L-shaped conformation. [ABSTRACT FROM AUTHOR]

An antimicrobial protein from seeds of Pharbitis nil (Pn-AMP) which shows an antifungal activity towards several agriculturally important plant pathogens has been crystallized in the presence of equimolar N-acetylglucosamine with sodium citrate as precipitant. The crystal belongs to the hexagonal space group P6122 (or P6522), with unit-cell parameters a = b = 29.33 (5), c = 133.44 (12) Å. Native data were collected using a crystal at 100 K to a resolution of 1.78 Å. [ABSTRACT FROM AUTHOR]

Ribosome recycling factor (RRF) catalyzes the disassembly of a termination complex during the final stage of protein synthesis. RRF from Escherichia coil has been crystallized with PEG 400 as precipitant at 287 K. The crystal belongs to the trigonal space group P3121 (or P3221), with unit-cell parameters a = b = 48.08, c= 141.67 Å. Native data were collected from a frozen crystal to a resolution of 3.0 Å on a Cu Kα rotating-anode X-ray source. [ABSTRACT FROM AUTHOR]

The transcription initiation factor I (TIF-IA) is an important regulator of the synthesis of ribosomal RNA (rRNA) through its facilitation of the recruitment of RNA polymerase I (Pol I) to the ribosomal DNA promoter. Activation of the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway, which occurs commonly in acute myelogenous leukemia, enhances rRNA synthesis through TIF-IA stabilization and phosphorylation. We have discovered that TIF-IA coexists with a splicing isoform, TIF-90, which is expressed preferentially in the nucleolus and at higher levels in proliferating and transformed hematopoietic cells. TIF-90 interacts directly with Pol I to increase rRNA synthesis as a consequence of Akt activation. Furthermore, TIF-90 binds preferentially to a 90-kDa cleavage product of the actin binding protein filamin A (FLNA) that inhibits rRNA synthesis. Increased expression of TIF-90 overcomes the inhibitory effect of this cleavage product and stimulates rRNA synthesis. Because activated Akt also reduces FLNA cleavage, these results indicate that activated Akt and TIF-90 function in parallel to increase rRNA synthesis and, as a consequence, cell proliferation in leukemic cells. These results provide evidence that the direct targeting of Akt would be an effective therapy in acute leukemias in which Akt is activated. [ABSTRACT FROM AUTHOR]

Bacterial pathogens have evolved diverse types of efficient machinery to acquire haem, the most abundant source of iron in the human body, and degrade it for the utilization of iron. Gram-positive bacteria commonly encode IsdG-family proteins as haem-degrading monooxygenases. Listeria monocytogenes is predicted to possess an IsdG-type protein (Lmo2213), but the residues involved in haem monooxygenase activity are not well conserved and there is an extra N-terminal domain in Lmo2213. Therefore, its function and mechanism of action cannot be predicted. In this study, the crystal structure of Lmo2213 was determined at 1.75 Å resolution and its haembinding and haem-degradation activities were confirmed. Structure-based mutational and functional assays of this protein, designated as an Isd-type L. monocytogenes haemdegrading enzyme (Isd-LmHde), identified that Glu71, Tyr87 and Trp129 play important roles in haem degradation and that the N-terminal domain is also critical for its haem-degrading activity. The haem-degradation product of Isd-LmHde is verified to be biliverdin, which is also known to be the degradation product of other bacterial haem oxygenases. This study, the first structural and functional report of the haemdegradation system in L. monocytogenes, sheds light on the concealed haem-utilization system in this life-threatening human pathogen. [ABSTRACT FROM AUTHOR]

Escherichia coli SdiA is a quorum-sensing (QS) receptor that responds to autoinducers produced by other bacterial species to control cell division and virulence. Crystal structures reveal that E. coli SdiA, which is composed of an N-terminal ligandbinding domain and a C-terminal DNA-binding domain (DBD), forms a symmetrical dimer. Although each domain shows structural similarity to other QS receptors, SdiA differs from them in the relative orientation of the two domains, suggesting that its ligand-binding and DNA-binding functions are independent. Consistently, in DNA gel-shift assays the binding affinity of SdiA for the ftsQP2 promoter appeared to be insensitive to the presence of autoinducers. These results suggest that autoinducers increase the functionality of SdiA by enhancing the protein stability rather than by directly affecting the DNA-binding affinity. Structural analyses of the ligand-binding pocket showed that SdiA cannot accommodate ligands with long acyl chains, which was corroborated by isothermal titration calorimetry and thermal stability analyses. The formation of an intersubunit disulfide bond that might be relevant to modulation of the DNA-binding activity was predicted from the proximal position of two Cys residues in the DBDs of dimeric SdiA. It was confirmed that the binding affinity of SdiA for the uvrY promoter was reduced under oxidizing conditions, which suggested the possibility of regulation of SdiA by multiple independent signals such as quorum-sensing inducers and the oxidation state of the cell. [ABSTRACT FROM AUTHOR]

The potential for pluripotent cells to differentiate into diverse specialized cell types has given much hope to the field of regenerative medicine. Nevertheless, the low efficiency of cell commitment has been a major bottleneck in this field. Here we provide a strategy to enhance the efficiency of early differentiation of pluripotent cells. We hypothesized that the initial phase of differentiation can be enhanced if the transcriptional activity of master regulators of stemness is suppressed, blocking the formation of functional transcriptomes. However, an obstacle is the lack of an efficient strategy to block protein-protein interactions. In this work, we take advantage of the biochemical property of seventeen kilodalton protein (Skp), a bacterial molecular chaperone that binds directly to sex determining region Y-box 2 (Sox2). The small angle X-ray scattering analyses provided a low resolution model of the complex and suggested that the transactivation domain of Sox2 is probably wrapped in a cleft on Skp trimer. Upon the transduction of Skp into pluripotent cells, the transcriptional activity of Sox2 was inhibited and the expression of Sox2 and octamer-binding transcription factor 4 was reduced, which resulted in the expression of early differentiation markers and appearance of early neuronal and cardiac progenitors. These results suggest that the initial stage of differentiation can be accelerated by inhibiting master transcription factors of stemness. This strategy can possibly be applied to increase the efficiency of stem cell differentiation into various cell types and also provides a clue to understanding the mechanism of early differentiation. [ABSTRACT FROM AUTHOR]

The DNA-dependent activator of IFN-regulatory factors (DAI), also known as DLM-1/ZBP1, initiates an innate immune response by binding to foreign DNAs in the cytosol. For full activation of the immune response, three DNA binding domains at the N terminus are required: two Z-DNA binding domains (ZBDs), Zcc and Zβ, and an adjacent putative B-DNA binding domain. The crystal structure of the Zβ domain of human DAI (hZβDAI) in complex with Z-DNA revealed structural features distinct from other known Z-DNA binding proteins, and it was classified as a group II ZBD. To gain structural insights into the DNA binding mechanism of hZβDAI, the solution structure of the free hZβDAI was solved, and its bindings to Band Z-DNAs were analyzed by NMR spectroscopy. Compared to the Z-DNA-bound structure, the conformation of free hZβDAI has notable alterations in the α3 recognition helix, the "wing." and Y145. which are critical in Z-DNA recognition. Unlike some other Zα domains. hZβDAI appears to have conformational flexibility, and structural adaptation is required for Z-DNA binding. Chemical-shift perturbation experiments revealed that hZβDAI also binds weakly to B-DNA via a different binding mode. The C-terminal domain of DAI is reported to undergo a conformational change on B-DNA binding; thus, it is possible that these changes are correlated. During the innate immune response, hZβDAI is likely to play an active role in binding to DNAs in both B and Z conformations in the recognition of foreign DNAs. [ABSTRACT FROM AUTHOR]

The receptor activator of nuclear factor-κB (RANK) and its ligand RANKL which belong to the tumor necrosis factor (TNF) receptor-ligand family, mediate osteoclastogenesis. The crystal structure of the RANKL ectodomain (eRANKL) in complex with the RANK ectodomain (eRANK) combined with biochemical assays of RANK mutants indicated that three RANK loops (Loop1, Loop2, and Loop3) bind to the interface of a trimeric eRANKL. Loop3 is particularly notable in that it is structurally distinctive from other TNF-family receptors and forms extensive contacts with RANKL The disulfide bond (C125-C127) at the tip of Loop3 is important for determining the unique topology of Loop3, and docking E126 close to RANKL which was supported by the inability of C127A or E126A mutants of RANK to bind to RANKL Inhibitory activity of RANK mutants, which contain loops of osteoprotegerin (OPG), a soluble decoy eceptor to RANKL, confirmed that OPG shares the similar binding mode with RANK and OPG. Loop3 plays a key role in RANKL binding. Peptide inhibitors designed to mimic Loop3 blocked the RANKL-induced differentiation of osteoclast precursors, suggesting that they could be developed as therapeutic agents for the treatment of osteoporosis and bonerelated diseases. Furthermore, some of the RANK mutations associated with autosomal recessive osteopetrosis (ARO) resulted in reduced RANKL-binding activity and failure to induce osteoclastogenesis. These results, together with structural interpretation of eRANK- eRANKL interaction, provided molecular understanding for pathogenesis of ARO. [ABSTRACT FROM AUTHOR]

The human RNA editing enzyme ADAR1 (double-stranded RNA deaminase I) deaminates adenine in pre-mRNA to yield inosine, which codes as guanine. ADAR1 has two left-handed Z-DNA binding domains, Zα and Zβ, at its NH2-terminus and preferentially binds Z-DNA, rather than B-DNA, with high binding affinity. The cocrystal structure of ZαADAR1 complexed to Z-DNA showed that one monomeric ZαADAR1 domain binds to one strand of double-stranded DNA and a second ZαADAR1 monomer binds to the opposite strand with 2-fold symmetry with respect to DNA helical axis. It remains unclear how ZαADAR1 protein specifically recognizes Z-DNA sequence in a sea of B-DNA to produce the stable ZαADAR1-Z-DNA complex during the B-Z transition induced by ZαADAR1. In order to characterize the molecular recognition of Z-DNA by ZαADAR1, we performed circular dichroism (CD) and NMR experiments with complexes of ZαADAR1 bound to d(CGCGCG)2 (referred to as CG6) produced at a variety of protein-to-DNA molar ratios. From this study, we identified the intermediate states of the CG6-αADAR1 complex and calculated their relative populations as a function of the ZαADAR1 concentration. These findings support an active B-Z transition mechanism in which the ZαADAR1 protein first binds to B-DNA and then converts it to left-handed Z-DNA, a conformation that is then stabilized by the additional binding of a second ZαADAR1 molecule. [ABSTRACT FROM AUTHOR]

The Escherichia coli envelope stress response is controlled by the alternative sigma factor, σ[supE], and is induced when unfolded outer membrane proteins accumulate in the periplasm. The response is initiated by sequential cleavage of the membrane-spanning antisigma factor, RseA. RseB is an important negative regulator of envelope stress response that exerts its negative effects on σ[supE] activity through its binding to RseA. In this study, we analyze the interaction between RseA and RseB. We found that tight binding of RseB to RseA required intact RseB. Using programs that performed global and local sequence alignment of RseB and RseA, we found regions of high similarity and performed alanine substitution mutagenesis to test the hypothesis that these regions were functionally important. This protocol is based on the hypothesis that functionally dependent regions of two proteins co-evolve and therefore are likely to be sequentially conserved. This procedure allowed us to identify both an N-terminal and C-terminal region in RseB important for binding to RseA. We extensively analyzed the C-terminal region, which aligns with a region of RseA coincident with the major RseB binding determinant in RseA. Both allele-specific suppression analysis and cysteine-mediated disuffide bond formation indicated that this C-terminal region of similarity of RseA and RseB identifies a contact site between the two proteins. We suggest a similar protocol can be successfully applied to pairs of non-homologous but functionally linked proteins to find specific regions of the protein sequences that are important for establishing functional linkage. [ABSTRACT FROM AUTHOR]

Mammalian DAI (DNA-dependent activator of IFN-regulatory factors), an activator of the innate immune response, senses cytosolic DNA by using 2 N-terminal Z-DNA binding domains (ZBDs) and a third putative DNA binding domain located next to the second ZBD. Compared with other previously known ZBDs, the second ZBD of human DAI (hZβDAI) shows significant variation in the sequence of the residues that are essential for DNA binding. In this article, the crystal structure of the hZβDAI/Z-DNA complex reveals that hZβDAI has a similar fold to that of other ZBDs, but adopts an unusual binding mode for recognition of Z-DNA. A residue in the first β-strand rather than residues in the β-loop contributes to DNA binding, and part of the (α3) recognition helix adopts a 310 helix conformation. The role of each residue that makes contact with DNA was confirmed by mutational analysis. The 2 ZBDs of DAI can together bind to DNA and both are necessary for full B-to-Z conversion. It is possible that binding 2 DAIs to 1 dsDNA brings about dimerization of DAI that might facilitate DNA-mediated innate immune activation. [ABSTRACT FROM AUTHOR]

The bacterial envelope stress response, which is responsible for sensing stress signals in the envelope and for turning on the σE-dependent transcription, is modulated by the binding of RseB to RseA. In this study, the solution structures of RseA and its complex with RseB were analyzed using circular dichroism and small-angle X-ray scattering. The periplasmic domain of RseA is unstructured and flexible when it is not bound to RseB. However, upon the formation of the stable complex with RseB, RseA induces conformational changes in RseB and, at the same time, RseA becomes more structured. Furthermore, it appears that some other undefined region of RseA, as well as the previously identified minimum region (amino acid 169-186), is also involved in RseB binding, It is thought that these conformational changes are relevant to the proteolytic cleavage of RseA and the modulation of envelope stress response. [ABSTRACT FROM AUTHOR]

The bacterial envelope stress response senses stress signals in the extracytoplasmic compartment, and activates σE-dependent transcription by degrading its antisigma factor RseA. RseB, a binding partner of RseA, plays a pivotal role in regulating this response, but its molecular mechanism is not understood. We therefore determined the crystal structure of Escherichia coli RseB at a resolution of 2.4 Å. RseB is composed of two domains linked by a flexible linker and forms a loosely packed dimer with two grooves on each side. This structural feature is confirmed by small-angle scattering in solution. Analysis of the binding of various RseA mutants to RseB allowed us to identify the major RseB-binding motif in RseA. These data, coupled with analysis of small-angle scattering of the RseA/RseB complex in solution, leads us to propose that two RseAs bind to the grooves of the dimeric RseB by conserved residues. The implications for modulating proteolytic cleavage of RseA are discussed. [ABSTRACT FROM AUTHOR]

Inorganic pyrophosphatase (PPase) is a ubiquitous cytosolic enzyme which catalyzes the hydrolysis of inorganic pyrophosphate (PPi) to orthophosphate (Pi). The crystal structure of inorganic pyrophosphatase from Helicobacter pylori (H-­PPase) has been solved by MAD and refined to an R factor of 20.6% at 2.6 Å resolution. The crystallographic asymmetric unit contains a homohexameric H-PPase arranged as a dimer of trimers. While most of the structural elements of PPases are highly conserved in H-PPase, some unique structural features are localized in the flexible loops near the active site, suggesting that the structural flexibility of these loops is required for the catalytic efficiency of PPase. [ABSTRACT FROM AUTHOR]

Left-handed Z-DNA is a higher-energy form of the double helix, stabilized by negative supercoiling generated by transcription or unwrapping nucleosomes. Regions near the transcription start site frequently contain sequence motifs favourable for forming Z-DNA, and formation of Z-DNA near the promoter region stimulates transcription. Z-DNA is also stabilized by specific protein binding; several proteins have been identified with low nanomolar binding constants. Z-DNA occurs in a dynamic state, forming as a result of physiological processes then relaxing to the right-handed B-DNA. Each time a DNA segment turns into Z-DNA, two B–Z junctions form. These have been examined extensively, but their structure was unknown. Here we describe the structure of a B–Z junction as revealed by X-ray crystallography at 2.6 Å resolution. A 15-base-pair segment of DNA is stabilized at one end in the Z conformation by Z-DNA binding proteins, while the other end remains B-DNA. Continuous stacking of bases between B-DNA and Z-DNA segments is found, with the breaking of one base pair at the junction and extrusion of the bases on each side (Fig. 1). These extruded bases may be sites for DNA modification. [ABSTRACT FROM AUTHOR]

Cephalosporin acylase is a member of the N-terminal hydrolase family, which is activated from an inactive precursor by autoproteolytic processing to generate a new N-terminal nucleophile Ser or Thr. The gene structure of the precursor cephalosporin acylases generally consists of a signal peptide that is followed by an α-subunit, a spacer sequence, and a β-subunit. The cephalosporin acylase precursor is post-translationally modified into an active heterodimeric enzyme with α- and β-subunits, first by intramolecular cleavage and, second, by intermolecular cleavage. Intramolecular autocatalytic proteolysis is initiated by nucleophilic attack of the residue Ser-1β onto the adjacent scissile carbonyl carbon. This study determined the precursor structure after disabling the intramolecular cleavage. This study also prowides experimental evidence showing that a conserved water molecule plays an important role in assisting the polarization of the OG atom of Set-1β to generate a strong nucleophile and to direct the OG atom of the Set-1β to a target carbonyl carbon. Intramolecular proteolysis is disabled as a result of a mutation of the residues causing conformational distortion to the active site. This is because distortion affects the existence of the catalytically crucial water at the proper position. This study provides the first evidence showing that a bound water molecule plays a critical role in initiating intramolecular cleavage in the post-translational modification of the precursor enzyme. [ABSTRACT FROM AUTHOR]

The small heat-shock protein (sHSP) from Methanococcus jannaschii(Mj HSP16.5) forms a homomeric complex of 24 subunits and has an overall structure of a multiwindowed hollow sphere with an external diameter of ≈120 Å and an internal diameter of ≈65 Å with six square "windows" of ≈17 Å across and eight triangular windows of ≈30 Å across. This sHSP has been known to protect other proteins from thermal denaturation. Using purified singlechain monellin as a substrate and a series of methods such as protease digestion, antibody binding, and electron microscopy, we show that the substrates bind to Mj HSP16.5 at a high temperature (80°C) on the outside surface of the sphere and are prevented from forming insoluble substrate aggregates in vitro. Circular dichroism studies suggest that a very small, if any, conformational change occurs in sHSP even at 80°C, but substantial conformational changes of the substrate are required for complex formation at 80°C. Furthermore, deletion mutation studies of Mj HSP16.5 suggest that the N-terminal region of the protein has no structural role but may play an important kinetic role in the assembly of the sphere by "preassembly condensation" of multiple monomers before final assembly of the sphere. [ABSTRACT FROM AUTHOR]

Investigates the mechanism of functional switch to protease by determining the crystal structure of the NH[sub 2]-terminal protease domain of high temperature requirement A. Indications that the activation of the proteolytic function of HtrA at elevated temperatures might occur by a conformational change; Biochemical activities of Tm HtrA.

α-Amylases (α-l,4-glucan-4-glucanohydrolase, E.C.3.2.1.1) catalyze the cleavage of α-I, 4-glucosidic linkages of starch components, glycogen, and various oligo saccharides. Thermostable α-amylases from Bacillus species are of great industrial importance in the production of corn syrup or dextrose. Thermostable α-amylase from Bacillus licheniformis, a monomeric enzyme with molecular mass of 55,200 Da (483 amino acid residues), shows a remarkable heat stability. This enzyme provides an attractive model for investigating the structural basis for thermostability of proteins. The three-dimensional structure of thermostable α-amylase from Bacillus licheniformis has been determined by the multiple isomorphous replacement method of X-ray crystallography. The structure has been refined to a crystallographic R-factor of 19.9% for 58,601 independent reflections with Fo>2σFo between 8.0 and 1.7 Å Resolution, with root mean square deviations of 0.013 A from ideal bond lengths and 1.720 from ideal bond angles. The final model consists of 469 amino acid residues and 294 water molecules. Missing from the model are the N- and C-termini and the segment between Trp182 and Asn192. Like other α-amylases, the polypeptide chain folds into three distinct domains. The first domain (domain A), consisting of 291 residues (from residue 3 to 103 and 207 to 396), forms a (β/a)8-barrel structure. The second domain (domain B), consisting of residues 104 to 206, is inserted between the third β-strand and the third α-helix of domain A. The third C-terminal domain (domain C), consisting of residues 397 to 482, folds into an eight-stranded antiparallel β-barrel. Neither calcium ion nor chloride ion is located near the active site. This study reveals the architecture of the thermostable α-amylase from Bacillus licheniformis. By homology with other α-amylases, important active site residues can be identified as Asp231, Glu261, and Asp328, which are all located at the C-terminal end of the central (β/a)8-barrel. Since many of the stabilizing and destabilizing mutations obtained so far fall in domain B or at its border, this region of the enzyme appears to be important for thermostability. The factors responsible for the remarkable thermostability of this enzyme may be increased ionic interactions, reduced surface area, and increased packing interactions in the interior. [ABSTRACT FROM AUTHOR]

The haloacid dehalogenase (HAD) protein superfamily is one of the largest enzyme families and shows hydrolytic activity towards diverse substrates. Structural analyses of enzymes belonging to the HAD family are required to elucidate the molecular basis underlying their broad substrate specificity and reaction mechanism. For this purpose, TON_1713, a hypothetical protein from Thermococcus onnurineus that is a member of the HAD superfamily, was expressed in Escherichia coli, purified and crystallized at 295 K using 1.6 M magnesium sulfate as a precipitant. X-ray diffraction data were collected to 1.8 Å resolution using a synchrotron-radiation source. The crystals belong to the triclinic space group P1, with unit-cell parameters a = 52.5, b = 65.8, c = 203.4 Å, α = 71.1, β = 79.9, γ = 74.3°. [ABSTRACT FROM AUTHOR]

Ketoprofen, a nonsteroidal anti-inflammatory drug, inhibits the synthesis of prostaglandin. A novel hydrolase (Est25) with high ketoprofen specificity has previously been identified using a metagenomic library from environmental samples. Recombinant Est25 protein with a histidine tag at the N-terminus was expressed in Escherichia coli and purified in a homogenous form. Est25 was crystallized from 2.4 M sodium malonate pH 7.0 and X-ray diffraction data were collected to 1.49 Å using synchrotron radiation. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 197.8, b = 95.2, c = 99.4 Å, β = 97.1°. [ABSTRACT FROM AUTHOR]

UDP-glucose pyrophosphorylase (UGPase) catalyzes the synthesis of UDP-glucose, an essential metabolite in all living organisms. An X-ray crystallographic study of UGPase from Helicobacter pylori has been performed in order to elucidate its role in the regulation of this important metabolic pathway. UGPase was crystallized from 0.1 M sodium acetate trihydrate pH 4.6, 2.0 M ammonium sulfate and 0.1 M guanidine-HCl. According to diffraction data collected at a resolution of 2.9 Å using a synchrotron-radiation source, the crystal belongs to the orthorhombic space group P212121, with unit-cell parameters a = 91.47, b = 98.61, c = 245.70 Å, α =β = γ = 90.0°. [ABSTRACT FROM AUTHOR]

CIpX, a member of the HSP (heat-shock protein) 100 family, functions as a molecular chaperone and is a regulatory subunit of the CIpXP protease. To understand the chaperone and regulatory mechanisms of CIpX, Helicobacter pylori CIpX has been over-expressed in Escherichia coli and crystallized at 295 K using (NH[SUB4])[SUB2]HPO[SUB4] as precipitant, X-ray diffraction data have been collected to 2.6Å resolution using a synchrotron-radiation source. The crystals belong to the hexagonal space group P6[SUB5] or P6[SUB1], with unit-cell parameters a = b = 78.52(04), c = 131.51(09) Å, α = &beta = 90, γ = 120°. The crystallographic asymmetric unit contains one molecule of CIpX, with a corresponding V[SUBM] of 2.78Å[SUB3] Da[SUP-1] and a solvent content of 55.8%. [ABSTRACT FROM AUTHOR]