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BBA - Proteins and Proteomics (v.1747, #2)
PA-I lectin from Pseudomonas aeruginosa binds acyl homoserine lactones by Raina N. Boteva; Vanya P. Bogoeva; Stoyanka R. Stoitsova (pp. 143-149).
The study analyses the binding affinities of Pseudomonas aeruginosa PA-I lectin (PA-IL) to three N-acyl homoserine lactones (AHSL), quorum sensing signal molecules responsible for cell–cell communication in bacteria. It shows that like some plant lectins, PA-IL has a dual function and, besides its carbohydrate-binding capacity, can accommodate AHLS. Formation of complexes between PA-IL and AHSL with acyl side chains composed of 4, 6 or 12 methyl groups is characterized by changes in the emissions of two incorporated fluorescent markers, TNS and IAEDANS, both derivatives of naphthalene sulfonic acid. PA-IL shows increasing affinities to lactones with longer aliphatic side chains. The values of the apparent dissociation constants ( Kd), which are similar to the previously determined Kd for the adenine high affinity binding, and the similar effects of lactones and adenine on the TNS emission indicate one identical binding site for these ligands, which is suggested to represent the central cavity of the oligomeric molecule formed after the association of the four identical subunits of PA-IL. Intramolecular distances between the fluorescent markers and protein Trp residues are determined by fluorescence resonance energy transfer (FRET).
Keywords: Lectin; Pseudomonas aeruginosa; TNS; IAEDANS; Fluorescence; FRET
Proteome analysis of mouse macrophages treated with anthrax lethal toxin by Harish Chandra; Pradeep K. Gupta; Kirti Sharma; Abid R. Mattoo; Satyendra K. Garg; W.N. Gade; Ravi Sirdeshmukh; Kapil Maithal; Yogendra Singh (pp. 151-159).
Anthrax toxin produced by Bacillus anthracis is a tripartite toxin comprising of protective antigen (PA), lethal factor (LF) and edema factor (EF). PA is the receptor-binding component, which facilitates the entry of LF or EF into the cytosol. EF is a calmodulin-dependent adenylate cyclase that causes edema whereas LF is a zinc metalloprotease and leads to necrosis of macrophages. It is also important to note that the exact mechanism of LF action is still unclear. With this view in mind, in the present study, we investigated a proteome wide effect of anthrax lethal toxin (LT) on mouse macrophage cells (J774A.1). Proteome analysis of LT-treated and control macrophages revealed 41 differentially expressed protein spots, among which phosphoglycerate kinase I, enolase I, ATP synthase (β subunit), tubulin β2, γ-actin, Hsp70, 14-3-3 zeta protein and tyrosine/tryptophan-3-monooxygenase were found to be down-regulated, while T-complex protein-1, vimentin, ERp29 and GRP78 were found to be up-regulated in the LT-treated macrophages. Analysis of up- and down-regulated proteins revealed that primarily the stress response and energy generation proteins play an important role in the LT-mediated macrophage cell death.
Keywords: Lethal toxin; Bacillus anthracis; Proteomics; Stress; ATP; Cytoskeletal
Identification and characterization of novel endogenous proteolytic forms of the human angiogenesis inhibitors restin and endostatin by Harald John; Kerstin Radtke; Ludger Ständker; Wolf-Georg Forssmann (pp. 161-170).
Restin and endostatin are C-terminal fragments of the noncollagenous domains of collagen XV and collagen XVIII exhibiting high sequence homology. Both polypeptides are distinguished by strong anti-angiogenic activity in vivo restricting the growth of solid tumors and metastasis. They are therefore currently being tested in clinical trials as anti-cancer drugs. We present the identification of new endogenous variants of both angiogenesis inhibitors isolated from a human hemofiltrate peptide library. Using an immunological screening approach with time-resolved rare earth metal fluorometry, immunoreactive compounds were purified chromatographically and characterized by mass spectrometry. We discovered four novel proteolytic products of restin as well as four variants of endostatin. Two endostatin products were characterized as short internal fragments (R176–L215 and R176–S219) of the entire molecule containing the recently identified β1 integrin receptor binding site, which plays a major role in endothelial cell migration and angiogenesis. Two additional forms contain mucin-type O-glycosylations. The O-glycosylated variants possess an oligosaccharide unit consisting of one N-acetylgalactosamine (GalNAc), one N-acetylneuraminic acid (NANA) and two galactose residues (Gal) occurring as sialo-(V117–S311–GalNAc–Gal2–NANA) and asialoglycopeptides (V117–S311–GalNAc–Gal2). The four restin variants (RI-RIV) were identified with identical C- but different N-termini and no posttranslational modification (RI: P66–A254, RII: P75–A254, RIII: Y81–A254 and RIV: A89–A254). Following a differential peptide mass fingerprint approach by reflector mode MALDI-TOFMS, the disulfide patterns of these circulating restins were determined as Cys1–Cys4 and Cys2–Cys3. These endogenous circulating collagen fragments will help to understand the physiological processing of the therapeutic proteins.
Keywords: Restin; Endostatin; Disulfide bond; Hemofiltrate; Peptide mass fingerprint; Mass spectrometry
Preparation and characterization of a single-chain calcineurin–calmodulin complex by Yunlong Qin; Jing Liu; Xin Li; Qun Wei (pp. 171-178).
Calcineurin (CN), a Ca2+/calmodulin (CaM)-dependent serine/threonine protein phosphatase, is a heterodimer composed of a catalytic subunit (CNA) and a regulatory subunit (CNB). The activity of CNA is under the control of two functionally distinct, but structurally similar Ca2+-regulated proteins, CaM and CNB. The crystal structure of the holoenzyme reveals that the N-terminus and C-terminus of CNB and the N-terminus of CNA each have a long arm not involved in the active site. We constructed a fusion of the genes of CaM, CNB and CNA in that order using linker primers containing six and ten codons of glycine. A single-chain CaM–CNB–CNA (CBA) complex was expressed and purified to near homogeneity. The single-chain complex was fully soluble, and had biochemical properties and kinetic parameters similar to single-chain CNB–CNA (BA) activated by CaM. It was not regulated by CaM and CNB, but was strongly stimulated by Mn2+, Ni2+ and Mg2+. Intrinsic fluorescence spectroscopy of the complex showed a change in the environment of tryptophan in the presence of Ca2+ and circular dichroism (CD) spectropolarimetry revealed an increase in alpha-helical content. Our findings suggest that fusion of CaM, CNB and CNA does not prevent the structural changes required for their functioning; in particular, CaM within the complex could still interact correctly with CN in the presence of Ca2+.
Keywords: Abbreviations; CN; calcineurin; CNA; calcineurin A subunit; CNB; calcineurin B subunit; CaM; calmodulin; CBA; CaM–CNB–CNA (single-chain calcineurin–calmodulin complex); BA; CNB–CNA (single-chain calcineurin); pNPP; p; -nitrophenyl phosphate; FPLC; fast protein liquid chromatography; CD; circular dichroismCalcineurin; Calmodulin; Fusion; Single-chain complex; Phosphatase activity; Interaction
Simultaneous refolding/purification of xylanase with a microwave treated smart polymer by Ipsita Roy; Kalyani Mondal; Aparna Sharma; Munishwar N. Gupta (pp. 179-187).
Affinity precipitation with a smart polymer, Eudragit S-100 (a methyl methacrylate polymer), was exploited for simultaneous refolding and purification of xylanase. Affinity precipitation consisted of this reversibly soluble–insoluble polymer-binding xylanase selectively. The complex was precipitated by lowering the pH and xylanase was eluted off the polymer using 1 M NaCl. For refolding experiments, the commercial preparation of Aspergillus niger xylanase was denatured with 8 M urea. Addition of microwave irradiated Eudragit S-100 and affinity precipitation led to recovery of 96% enzyme activity by refolding. Simultaneously, the enzyme was purified 45 times. Thermally inactivated preparation, when subjected to similar steps, led to 95% recovery of enzyme activity with 42-fold purification. The strategy has the potential for recovering pure proteins in active forms from overexpressed proteins, which generally form inclusion bodies in E. coli.
Keywords: Affinity precipitation; Inclusion body; Microwave treatment; Protein refolding; Thermal denaturation; Urea denaturation; Xylanase
Echinococcus granulosus antigen B hydrophobic ligand binding properties by Gustavo Chemale; Henrique B. Ferreira; John Barrett; Peter M. Brophy; Arnaldo Zaha (pp. 189-194).
Antigen B (AgB), an immunodominant component of the cestode parasite Echinococcus granulosus, presents homology to and shares apparent structural similarities with helix-rich hydrophobic ligand binding proteins (HLBPs) from other cestodes. In order to investigate the fatty acid binding properties of AgB, two of its subunit components (rAgB8/1 and rAgB8/2) were expressed in Escherichia coli and purified, and the native antigen was purified from the hydatid cyst fluid by affinity chromatography using a monoclonal antibody raised against rAgB8/1. The interaction of the purified native and recombinant proteins with the fluorescent ligands DAUDA, ANS, DACA and 16-AP was investigated. The palmitic acid derived fluorescent ligand, 16-AP, showed the greatest enhancement in fluorescence when bound to native AgB or to its recombinant subunits, and the dissociation constants for 16-AP binding were determined. Surprisingly, in contrast to HLBPs from other cestodes, interactions with other fatty acids, including palmitic acid, caused an increase in fluorescence instead of competing with 16-AP. Our results suggest that AgB might have evolved different functions in the binding of hydrophobic compounds, dependent on cestode environment.
Keywords: Abbreviations; AgB; antigen B; HLBP; hydrophobic ligand binding protein; HF; hydatid fluid; 16-AP; 16-(9-anthroyloxy) palmitate Echinococcus granulosus; Antigen B; Hydrophobic ligand binding protein; Fatty acid; 16-(9-Anthroyloxy) palmitate (16-AP)
Tyrosine B10 and heme–ligand interactions of Lucina pectinata hemoglobin II: control of heme reactivity by Ruth Pietri; Laura Granell; Anthony Cruz; Walleska De Jesús; Ariel Lewis; Ruth Leon; Carmen L. Cadilla; Juan López Garriga (pp. 195-203).
The distal pocket of hemoglobin II (HbII) from Lucina pectinata is characterized by the presence of a GlnE7 and a TyrB10. To elucidate the functional properties of HbII, biophysical studies were conducted on HbII and a HbI PheB10Tyr site-directed mutant. The pH titration data at neutral conditions showed visible bands at 486, 541, 577 and 605 nm for both proteins. This suggests the possible existence of a conformational equilibrium between an open and closed configuration due to the interactions of the TyrB10, ligand, and heme iron. The kinetic behavior for the reaction of both ferric proteins with H2O2 indicates that the rate for the formation of the ferryl intermediates species varies with pH, suggesting that the reaction is strongly dependent on the conformational states. At basic pH values, the barrier for the reaction increases as the tyrosine adopts a closed conformation and the ferric hydroxyl replaces the met-aquo species. The existence of these conformers is further supported by resonance Raman (RR) data, which indicate that in a neutral environment, the ferric HbII species is present as a possible mixture of coordination and spin states, with values at 1558 and 1580 cm−1 for the ν2 marker, and 1479, 1492, and 1503 cm−1 for the ν3 mode. Moreover, the presence of the A3 and Ao conformers at 1924 and 1964 cm−1 in the HbII-CO infrared spectra confirms the existence of an open and closed conformation due to the orientation of the TyrB10 with respect to the heme active center.
Keywords: Lucina pectinata; Hemoglobin II; Closed conformation; Open conformation; HbI PheB10Tyr mutant
Metal binding of metallothionein-3 versus metallothionein-2: lower affinity and higher plasticity by Peep Palumaa; Indrek Tammiste; Keiu Kruusel; Liina Kangur; Hans Jörnvall; Rannar Sillard (pp. 205-211).
Mammalian metallothioneins (MTs) are involved in cellular metabolism of zinc and copper and in cytoprotection against toxic metals and reactive oxygen species. MT-3 plays a specific role in the brain and is down-regulated in Alzheimer's disease. To evaluate differences in metal binding, we conducted direct metal competition experiments with MT-3 and MT-2 using electrospray ionization mass spectroscopy (ESI-MS). Results demonstrate that MT-3 binds Zn2+ and Cd2+ ions more weakly than MT-2 but exposes higher metal-binding capacity and plasticity. Titration with Cd2+ ions demonstrates that metal-binding affinities of individual clusters of MT-2 and MT-3 are decreasing in the following order: four-metal cluster of MT-2>three-metal cluster of MT-2≈four-metal cluster of MT-3>three-metal cluster of MT-3>extra metal-binding sites of MT-3. To evaluate the reasons for weaker metal-binding affinity of MT-3 and the enhanced resistance of MT-3 towards proteolysis under zinc-depleted cellular conditions, we studied the secondary structures of apo-MT-3 and apo-MT-2 by CD spectroscopy. Results showed that apo-MT-3 and apo-MT-2 have almost equal helical content (approximately 10%) in aqueous buffer, but that MT-3 had slightly higher tendency to form α-helical secondary structure in TFE–water mixtures. Secondary structure predictions also indicated some differences between MT-3 and MT-2, by predicting random coil for common MTs, but 22% α-helical structure for MT-3. Combined, all results highlight further differences between MT-3 and common MTs, which may be related with their functional specificities.
Keywords: Abbreviations; MT; metallothionein; DTT; dithiothreitol; TFE; 2,2,2,-trifluoroethanol; CD; circular dichroism; ESI MS; electrospray ionization mass spectroscopyMT-3; Alzheimer's disease; ESI-MS; Secondary structure prediction; CD spectroscopy
Taiwan cobra chymotrypsin inhibitor: cloning, functional expression and gene organization by Yun-Ching Cheng; Fang-Jiun Yan; Long-Sen Chang (pp. 213-220).
A cDNA encoding chymotrypsin inhibitor was constructed from the cellular RNA isolated from the venom glands of Naja atra (Taiwan cobra). The resultant amino acid sequence showed that the mature protein is comprised of 57 amino acid residues with six cysteine residues. Cloned protein was expressed and isolated from the inclusion bodies of E. coli and refolded into a functional protein in vitro. Deleting the first three residues at its N-terminus caused a moderate increase in the inhibitory constant ( Ki) against chymotrypsin. The genomic DNA encoding the chymotrypsin inhibitor was amplified by PCR. The gene shares virtually an identical structural organization with the β-bungarotoxin B1 chain (a snake Kunitz/BPTI neurotoxic homolog) gene. Moreover, the overall sequence identity of the N. atra chymotrypsin inhibitor and β-bungarotoxin B1 chain genes was up to 83%. These findings strongly suggest that snake Kunitz/BPTI protease inhibitors and neurotoxic homologs may have originated from a common ancestor.
Keywords: Chymotrypsin inhibitor; Mutagenesis of N-terminus; Exon–intron organization; Naja atra
Design, expression and characterisation of lysine-rich forms of the barley seed protein CI-2 by Jane L. Forsyth; Frederic Beaudoin; Nigel G. Halford; Richard B. Sessions; Anthony R. Clarke; Peter R. Shewry (pp. 221-227).
Chymotrypsin inhibitor CI-2 is a small (84 residue) barley seed protein that has been used extensively to study protein folding. It also contains eight lysine residues, making it an attractive target for expression in transgenic plants to increase their lysine contents. We have designed three lysine-enriched forms of CI-2 and compared their structures and properties with that of the wild type protein. One mutant containing three additional lysine residues in the inhibitory loop shows high stability to denaturation and reduced inhibitory activity, indicating its suitability for use in genetic engineering.
Keywords: Chymotrypsin inhibitor; Protein engineering; Lysine enhancement; Nutritional quality; Barley
Ligand-binding specificity of an invertebrate ( Manduca sexta) putative cellular retinoic acid binding protein by Claudia Folli; Ileana Ramazzina; Riccardo Percudani; Rodolfo Berni (pp. 229-237).
Intracellular lipid-binding proteins (iLBPs) are small cytoplasmic proteins that specifically interact with hydrophobic ligands. Fatty acid-binding proteins (FABPs), cellular retinoic acid-binding proteins (CRABPs) and cellular retinol-binding proteins (CRBPs) belong to the iLBP family. A recently identified insect ( Manduca sexta) iLBP has been reported to possibly represent an invertebrate CRABP mimicking the role of CRABPs in vertebrate organisms. The presence in this protein of the characteristic binding triad residues involved in the interaction with ligand carboxylate head groups, a feature pertaining to several FABPs and to CRABPs, and the close phylogenetic relationships with both groups of vertebrate heart-type FABPs and CRBPs/CRABPs, makes it difficult to assign it to either FABPs or CRABPs. However, its negligible interaction with retinoic acid and high affinity ( Kd values in the 10−8 M range) for fatty acids have been established by means of direct and competitive binding assays. As shown by phylogenetic analysis, the M. sexta iLBP belongs to a wide group of invertebrate iLBPs, which, besides being closely related phylogenetically, share distinctive features, such as the conservation of chemically distinct residues in their amino acid sequences and the ability to bind fatty acids. Our results are in keeping with the lack of cellular retinoid-binding proteins in invertebrates and with their later appearance during the course of chordate evolution.
Keywords: Abbreviations; ANS; 8-anilinonaphthalene-1-sulfonic acid; ALBP; adipocyte lipid-binding protein; B-FABP; brain fatty acid-binding protein; CRBP; cellular retinol-binding protein; CRABP; cellular retinoic acid-binding protein; E-FABP; epidermal fatty acid-binding protein; FABP; fatty acid-binding protein; H-FABP; heart fatty acid-binding protein; iLBP; intracellular lipid-binding protein; ILBP; ileal lipid-binding protein; I-FABP; intestinal fatty acid-binding protein; L-FABP; 1; liver fatty acid-binding protein; L-FABP; 2; liver basic fatty acid-binding protein; MLBP; myelin P2; TLBP; testicular lipid-binding proteinFatty acid-binding protein; Retinoic acid-binding protein; Retinoid; Ligand binding; Phylogenetic analysis; Insect binding protein
FMN binding and unfolding of Desulfovibrio desulfuricans flavodoxin: “hidden� intermediates at low denaturant concentrations by B.K. Muralidhara; Pernilla Wittung-Stafshede (pp. 239-250).
The flavin mononucleotide (FMN) cofactor in Desulfovibrio desulfuricans flavodoxin stays associated with the polypeptide upon guanidine hydrochloride (GuHCl) induced unfolding. Using isothermal titration calorimetry (ITC), we determined the affinity of FMN for the flavodoxin polypeptide as a function of both urea and GuHCl concentrations (pH 7, 25 °C). The FMN affinity for folded and GuHCl-unfolded flavodoxin differs 10-fold, which is in agreement with the difference in thermodynamic stability between the apo- and holo-forms. In contrast, the urea-unfolded protein does not interact with FMN and equilibrium unfolding of holo-flavodoxin in urea results in FMN dissociation prior to polypeptide unfolding. ANS-binding, near-UV circular dichroism (CD), acrylamide quenching and FMN-emission experiments reveal the presence of native-like intermediates, not detected by far-UV CD and aromatic fluorescence detection methods, in low concentrations of both denaturants. Time-resolved experiments show that FMN binding is fastest at GuHCl concentrations where the native-like intermediate species is populated.
Keywords: Abbreviations; CD; circular dichroism; FMN; flavin mononucleotide; ITC; isothermal titration calorimetry; K; D; dissociation constant; GuHCl; guanidine hydrochloride; ANS; 8-anilinonaphthalene-1sulfonic acidFlavin mononucleotide; Flavodoxin; Protein folding; Isothermal titration calorimetry; Stopped-flow mixing; Equilibrium unfolding
Bacterial expression, folding, purification and characterization of soluble NTPDase5 (CD39L4) ecto-nucleotidase by Deirdre M. Murphy-Piedmonte; Patrick A. Crawford; Terence L. Kirley (pp. 251-259).
The ecto-nucleoside triphosphate diphosphohydrolases (eNTPDases) are a family of enzymes that control the levels of extracellular nucleotides, thereby modulating purinergically controlled physiological processes. Six of the eight known NTPDases are membrane-bound enzymes; only NTPDase 5 and 6 can be released as soluble enzymes. Here we report the first bacterial expression and refolding of soluble human NTPDase5 from inclusion bodies. The results show that NTPDase5 requires the presence of divalent cations (Mg2+ or Ca2+) for activity. Positive cooperativity with respect to hydrolysis of its preferred substrates (GDP, IDP and UDP) is observed, and this positive cooperativity is attenuated in the presence of nucleoside monophosphate products (e.g., GMP and AMP). In addition, comparing the biochemical properties of wild-type NTPDase5 and those of a mutant NTPDase5 (C15S, which lacks the single, non-conserved cysteine residue), also expressed in bacteria, suggests that Cys15 is not essential for either proper refolding or enzymatic activity (indicating this residue is not involved in a disulfide bond). Moreover, the substrate profile of bacterially expressed NTPDase5, as well as the C15S mutant, was determined to be similar to that of full-length, membrane-bound and soluble NTPDase5 expressed in mammalian COS cells.
Keywords: Abbreviations; NTPDases; n; ucleoside; t; ri; p; hosphate; d; iphosphohydrol; ases; NTPDase5; nucleoside triphosphate diphosphohydrolase type 5; ACRs; apyrase conserved regions; B-PER; bacterial protein extraction reagent; MWCO; molecular weight cut off; P; i; inorganic phosphate; MOPS; 3-[; N; -morpholino]propanesulfonic acid; IPTG; isopropyl-β-; d; -thiogalactopyranoside; DTT; dithiothreitol; DMEM; Dulbecco's modified Eagle medium; NTA; nitrilotriacetic acid; KLH; keyhole limpet hemocyanin; TBS; Tris-buffered saline; SEC; size exclusion chromatographyNucleoside triphosphate diphosphohydrolase; NTPDase5; CD39L4; Bacterial expression; Protein refolding; Enzymatic characterization; Positive cooperativity
Comparison of cAMP-dependent protein kinase substrate specificity in reaction with proteins and synthetic peptides by Mart Loog; Nikita Oskolkov; Fergal O'Farrell; Pia Ek; Jaak Järv (pp. 261-266).
Mutants of L-type pyruvate kinase with modified peptide sequence around the Ser-12 phosphorylation site were prepared and kinetics of their phosphorylation by protein kinase A was studied. The profile of substrate specificity obtained for these proteins was compared with the kinetic data of phosphorylation of short peptide substrates. Alterations made in protein structure caused weaker effects than the corresponding alterations made in peptides, while the amino acid preferences and the overall specificity pattern remained similar in the both cases. Thus, similar consensus motif holds for both protein and peptide substrates, but is less critical for recognition of proteins if compared with short peptides.
Keywords: Abbreviations; PKA-cAMP; dependent protein kinase A catalytic subunit; L-PK; L-type pyruvate kinase; IPTG; isopropyl β-; d; -thiogalactopyranoside; SDS; sodium dodecyl sulfateProtein kinase A; Substrate specificity; Peptide; Protein; L-type pyruvate kinase; Phosphorylation
The sulfur oxygenase reductase from Acidianus ambivalens is an icosatetramer as shown by crystallization and Patterson analysis by Tim Urich; Ricardo Coelho; Arnulf Kletzin; Carlos Frazao (pp. 267-270).
The sulfur oxygenase reductase (SOR) is the initial enzyme in the aerobic sulfur metabolism of the thermoacidophilic and chemolithoautotrophic crenarchaeote Acidianus ambivalens. Single colorless polyhedral crystals were obtained under two crystallization conditions from SOR preparations heterologously overproduced in Escherichia coli. They belonged to space-group I4 and diffraction data were collected up to 1.7 Å resolution. Their Patterson symmetry shows additional 4-, 3- and 2-fold non-crystallographic symmetry rotation axes, characteristic of the point group 432. Taking into account the molecular mass of SOR, the crystal unit cell volume, the non-crystallographic symmetry operators and previous electron microscopy studies of the SOR, it was deduced that the quaternary structure of the functionally active enzyme is an icosatetramer with 871 kDa molecular mass.
Keywords: Crystallization; Non-heme iron protein; Sulfur metabolism; Archaea; Extremophile; Quaternary structure
First-time crystallization and preliminary X-ray crystallographic analysis of a bacterial-archaeal type UMP kinase, a key enzyme in microbial pyrimidine biosynthesis by Clara Marco-Marín; Juan Manuel Escamilla-Honrubia; Vicente Rubio (pp. 271-275).
UMP phosphorylation, a key step for pyrimidine nucleotide biosynthesis, is catalyzed in bacteria by UMP kinase (UMPK), an enzyme specific for UMP that is dissimilar to the eukaryotic UMP/CMP kinase or to other nucleoside monophosphate kinases. UMPK is allosterically regulated and participates in pyrimidine-triggered gene repression. As first step towards determining UMPK structure, the putative UMPK-encoding gene of the hyperthermophilic archaeon Pyrococcus furiosus was cloned and overexpressed in Escherichia coli. The protein product was purified and confirmed to be a genuine UMPK. It was crystallized at 294 K in hanging drops by the vapor diffusion technique using 3.5–4 M Na formate. Cubic 0.2-mm crystals diffracted synchrotron X-rays to 2.4-Å resolution. Space group was I23 ( a= b= c=144.95 Å), and the asymmetric unit contained two monomers, with 52% solvent content. The self-rotation function suggests that the enzyme is hexameric, which agrees with biochemical studies on bacterial UMPKs.
Keywords: Pyrimidine nucleotide biosynthesis; Amino acid kinase family; Uridylate kinase; UMP kinase