The hemK gene in Escherichia coli encodes the N(5)-glutamine methyltransferase that modifies peptide release factors

Class 1 peptide release factors (RFs) in Escherichia coli are N(5)-methylated on the glutamine residue of the universally conserved GGQ motif. One other protein alone has been shown to contain N(5)-methylglutamine: E.coli ribosomal protein L3. We identify the L3 methyltransferase as YfcB and show that it methylates ribosomes from a yfcB strain in vitro, but not RF1 or RF2. HemK, a close orthologue of YfcB, is shown to methylate RF1 and RF2 in vitro. hemK is immediately downstream of and co-expressed with prfA. Its deletion in E.coli K12 leads to very poor growth on rich media and abolishes methylation of RF1. The activity of unmethylated RF2 from K12 strains is extremely low due to the cumulative effects of threonine at position 246, in place of alanine or serine present in all other bacterial RFs, and the lack of N(5)-methylation of Gln252. Fast-growing spontaneous revertants in hemK K12 strains contain the mutations Thr246Ala or Thr246Ser in RF2. HemK and YfcB are the first identified methyltransferases modifying glutamine, and are widely distributed in nature.     

Crystallization and preliminary X-ray crystallographic analysis of UDP-N-acetylglucosamine enolpyruvyl transferase from Haemophilus influenzae in complex with UDP-N-acetylglucosamine and fosfomycin

The bacterial enzyme UDP-N-acetylglucosamine enolpyruvyl transferase catalyzes the first committed step of peptidoglycan biosynthesis, i.e., transfer of enolpyruvate from phosphoenolpyruvate to UDP-N-acetyl-glucosamine. We have overexpressed the enzyme from Haemophilus influenzae in Escherichia coli and crystallized it in the apo-form, as well as in a complex with UDP-N-acetylglucosamine and fosfomycin using ammonium sulfate as the precipitant. X-ray diffraction data from a crystal of the apo-form were collected to 2.8 A resolution at 293 K. The crystal quality was improved by co-crystallization with UDP-N-acetylglucosamine and fosfomycin. X-ray data to 2.2 A have been collected at 100 K from a flash-frozen crystal of the complex. The complex crystals belong to the orthorhombic space group I222 (or I212121) with unit-cell parameters of a = 63.7, b = 124.5, and c = 126.3 A. Assuming a monomer of the recombinant enzyme in the crystallographic asymmetric unit, the calculated Matthews parameter (VM) is 2.71 A3 Da-1 and solvent content is 54.6%.     

Crystallization and preliminary crystallographic study of DNA polymerase from Pyrococcus furiosus

A new member of archaeal DNA polymerase from Pyrococcus furiosus was crystallized. Diffraction data to 3.1 A of the selenomethionine-derivatized crystal were collected, and preliminary crystallographic study has been completed. The crystal belongs to the space group C2 with unit cell parameters of a = 93.2 A, b = 124.9 A, c = 87.7 A, alpha = 90 degrees , beta = 109.7 degrees , and gamma = 90 degrees . Assuming the presence of one molecule in the asymmetric unit, the solvent content of the crystal is estimated to be 54%, corresponding to a Matthews coefficient V(M) of 2.7A (3) Da(-1).     

Overexpression, crystallization, and preliminary X-ray crystallographic analysis of the alanine racemase from Enterococcus faecalis v583

Alanine racemase, a bacterial enzyme belonging to the fold-type III group of pyridoxal 5'-phosphate (PLP)-dependent enzymes, has been shown to catalyze the interconversion between L- and D-alanine. The alanine racemase from the pathogenic bacterium Enterococcus faecalis v583 has been overexpressed in E. coli and was shown to crystallize an enzyme at 295 K, using polyethylene glycol (PEG) 8000 as a precipitant. X-ray diffraction data to 2.5 A has been collected using synchrotron radiation. The crystal is a member of the orthorhombic space group, C222(1), with unit cell parameter of a=94.634, b=156.516, c=147.878 A, and alpha=beta;=gamma=90 degrees. Two or three monomers are likely to be present in the asymmetric unit, with a corresponding Vm of 3.38 A3 Da(-1) and 2.26 A Da(-1) and a solvent content of 63.7% and 45.5%, respectively.     

Crystallization and preliminary X-ray studies on Sulfolobus acidocaldarius ferredoxin.

Ferredoxin from a thermoacidophilic archaebacterium, Sulfolobus acidocaldarius, has been crystallized. The space group is P4(3)2(1)2 or P4(1)2(1)2 and the cell dimensions are a = b = 50.12 A and c = 69.52 A. The Vm value is calculated to be 1.88 A3/Da, assuming one molecule per asymmetric unit. The crystal diffracts X-rays beyond 2.0 A resolution.     

The type I/type II cytochrome c3 complex: an electron transfer link in the hydrogen-sulfate reduction pathway

In Desulfovibrio metabolism, periplasmic hydrogen oxidation is coupled to cytoplasmic sulfate reduction via transmembrane electron transfer complexes. Type II tetraheme cytochrome c3 (TpII-c3), nine-heme cytochrome c (9HcA) and 16-heme cytochrome c (HmcA) are periplasmic proteins associated to these membrane-bound redox complexes and exhibit analogous physiological function. Type I tetraheme cytochrome c3 (TpI-c3) is thought to act as a mediator for electron transfer from hydrogenase to these multihemic cytochromes. In the present work we have investigated Desulfovibrio africanus (Da) and Desulfovibrio vulgaris Hildenborough (DvH) TpI-c3/TpII-c3 complexes. Comparative kinetic experiments of Da TpI-c3 and TpII-c3 using electrochemistry confirm that TpI-c3 is much more efficient than TpII-c3 as an electron acceptor from hydrogenase (second order rate constant k = 9 x 10(8) M(-1) s(-1), K(m) = 0.5 microM as compared to k = 1.7 x 10(7) M(-1) s(-1), K(m) = 40 microM, for TpI-c3 and TpII-c3, respectively). The Da TpI-c3/TpII-c3 complex was characterized at low ionic strength by gel filtration, analytical ultracentrifugation and cross-linking experiments. The thermodynamic parameters were determined by isothermal calorimetry titrations. The formation of the complex is mainly driven by a positive entropy change (deltaS = 137(+/-7) J mol(-1) K(-1) and deltaH = 5.1(+/-1.3) kJ mol(-1)) and the value for the association constant is found to be (2.2(+/-0.5)) x 10(6) M(-1) at pH 5.5. Our thermodynamic results reveal that the net increase in enthalpy and entropy is dominantly produced by proton release in combination with water molecule exclusion. Electrostatic forces play an important role in stabilizing the complex between the two proteins, since no complex formation is detected at high ionic strength. The crystal structure of Da TpI-c3 has been solved at 1.5 angstroms resolution and structural models of the complex have been obtained by NMR and docking experiments. Similar experiments have been carried out on the DvH TpI-c3/TpII-c3 complex. In both complexes, heme IV of TpI-c3 faces heme I of TpII-c3 involving basic residues of TpI-c3 and acidic residues of TpII-c3. A secondary interacting site has been observed in the two complexes, involving heme II of Da TpII-c3 and heme III of DvH TpI-c3 giving rise to a TpI-c3/TpII-c3 molar ratio of 2:1 and 1:2 for Da and DvH complexes, respectively. The physiological significance of these alternative sites in multiheme cytochromes c is discussed.     

Crystallization and preliminary X-ray diffraction studies of pancreatic spasmolytic polypeptide.

Pancreatic spasmolytic polypeptide (PSP) isolated from porcine pancreas has been crystallized by the hanging drop vapour diffusion method. The crystals belong to the space group I222 or I2(1)2(1)2(1) with cell dimensions a = 181.9 A, b = 54.5 A, c = 72.9 A. The crystals diffract to at least 2.5 A resolution and the asymmetric unit contains two molecules (Vm = 3.9 A3/Da) with a solvent content of 68% as determined by density measurements of the crystals. The self-rotation function suggests that the two molecules within the asymmetric unit are related by a 2-fold axis at either 30 degrees or 60 degrees from a in a plane perpendicular to the b axis.     

Crystal structure of SCO6571 from Streptomyces coelicolor A3(2)

SCO6571 protein from Streptomyces coelicolor A3(2) was overexpressed and purified using Rhodococcus erythropolis as an expressing host. Crystals of selenomethionine-substituted SCO6571 have been obtained by vapor diffusion method. SCO6571 crystals diffract to 2.3 A and were found to belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell parameters a = 84.5, b = 171.6, c = 184.8 A. Six molecules in the asymmetric unit give a crystal volume per protein mass (V(M)) of 2.97 A (3) Da(-1) and solvent content of 58.6 %. The structure was solved by the single wavelength anomalous diffraction (SAD) method. SCO6571 is a TIM-barrel fold protein that assembles into a hexameric molecule with D(3) symmetry.     

Crystallization and preliminary X-ray diffraction studies of Streptomyces phospholipase A2 in a calcium-binding form

Phospholipase A2 (PLA2) as a calcium-binding form, produced by Streptomyces violaceoruber, was crystallized in a form suitable for the diffraction analysis using the vapor diffusion method. Crystals were grown in 0.1 M Tris-HCl buffer (pH 8.5), 20 mM Ca2+ containing 50-60% (v/v) 2-methyl-2,4-pentanediol as a precipitant. They belong to the monoclinic space group P2(1), with the cell dimensions a=38.3 A, b=54.3 A, c=30.6 A, and beta=90.2 degrees. The crystals diffract the X-ray well and the diffraction intensity data were collected up to 1.6 A resolution. The crystal volume per unit mass, V(M) is 2.35 A3 Da(-1) with one molecule in the asymmetric unit, which corresponds to a solvent content of 47.7%.     

The target of RNAIII-activating protein (TRAP) from Staphylococcus aureus: purification, crystallization and preliminary X-ray analysis

The target of the RNAIII-activating protein (TRAP) is a 21 kDa protein in which phosphorylation is activated by the RNAIII-activating protein (RAP), which causes an increase in RNAII and RNAIII synthesis and the production of the virulence factors. In an attempt to examine the structural role of TRAP in the signal transduction pathway, TRAP from Staphylococcus aureus was overexpressed, purified and crystallized using PEG 8000 and 5% Jeffamine M600 (pH 7.0), as precipitants by hanging-drop vapour diffusion methods at 287 K. The crystals belong to the orthorhombic space group, P2(1)2(1)2(1), with unit cell parameters of a=39.68, b=50.41, c=85.45 A. There is one monomer of TRAP per crystallographic asymmetric unit with a crystal volume per protein mass (V(M)) of 2.06 A(3) Da(-1) and a solvent content of 40.3%. A complete data set diffracting to 1.9 A resolution was collected from a single crystal at 100 K using a synchrotron-radiation source.     

M-S vibrational study in three-coordinate thiolato compounds (NEt4)2[M(SC6H4-p-X)3] and (NEt4)

By using p-substituted benzenethiolate ligands, the novel three-coordinate copper(I) and silver(I) thiolato complexes (NEt4)2[Cu(SC6H4-p-X)3] (X=Cl (1) and Br (2)), (NEt4)2[Ag(SC6H4-p-X)3] (X=Cl (3) and Br (4)) and novel clusters (NEt4)2[M4(mu-SC6H4-p-Cl)6] (M=Cu (5) and Ag(6)) have been prepared and structurally characterized by single crystal X-ray diffraction. All the complexes have three-coordinate sites having point-group D3h symmetry. The three-coordinate mononuclear silver(I) complexes 3 and 4 are the first examples. The M-S stretching bands were determined by far-IR and FT-Raman spectroscopies; nu(Cu-S) 363-372 cm(-1) and nu(Ag-S) 353-363 cm(-1). These results indicate that M-S stretching vibration energy in the three-coordinate metal(I) site of the mononuclear compounds or clusters is around 340-380 cm(-1), and it is a useful tool for determining their coordination modes.     

Purification, crystallization and preliminary X-ray studies of AxCesD required for efficient cellulose biosynthesis in Acetobacter xylinum

AxCesD protein required for bacterial cellulose biosynthesis in Acetobacter xylinum was overexpressed in E. coli, purified and crystallized. Single crystals of SeMet-substituted AxCesD were obtained by the sitting-drop vapor-diffusion method. The crystal belongs to the primitive trigonal space group P3 2, with unit-cell parameters a = b = 77.7 A, and c = 213.9 A. The asymmetric unit in the crystal was assumed to contain 8 protein molecules giving the Matthews coefficient (VM) of 2.54 A3 Da(-1). Se-MAD data were collected to 2.3 A resolution using synchrotron radiations.     

Crystallization and preliminary X-ray diffraction analysis of gingipain R2 from Porphyromonas gingivalis in complex with H-D-Phe-Phe-Arg-chloromethylketone.

Gingipain R2 is a 50 kDa proteinase from the oral pathogenic bacterium Porphyromonas gingivalis. This proteinase, which displays no significant sequence homology to any protein previously analyzed by X-ray crystallography, has been crystallized using the vapor diffusion method. Two different crystal forms were obtained from a solution containing polyethylene glycol (MW 8,000) (space group P2(1)2(1)2(1)) or magnesium sulfate (space group R3) as precipitating agent. Complete diffraction data sets have been collected up to 2.0 and 2.9 A resolution, respectively. Cell dimensions are a = 51.9 A, b = 79.9 A, and c = 99.6 A (P2(1)2(1)2(1)), and a = b = 176.6 A, and c = 143.4 A (R3). Considerations of the possible values of Vm accounts for the presence of one monomer per asymmetric unit in the case of the orthorhombic crystal form, whereas the rhombohedral crystal form, together with the analysis of the self-rotation function, could accommodate a tetramer in the asymmetric unit.     

Dopaminergic modulation of ventilation in obese Zucker rats.

To investigate the hypothesis that the impaired respiratory drive noted in morbid obesity was attributable to altered dopaminergic mechanisms acting on peripheral and/or central chemoreflex sensitivity, seven obese and seven lean Zucker rats were studied at 11 wk of age. Ventilation (VE) was measured by the barometric technique during hyperoxic (100% O(2)), normoxic (21% O(2)), hypoxic (10% O(2)), and hypercapnic (7% CO(2)) exposures after the administration of vehicle (control), haloperidol [Hal, 1 mg/kg, a central and peripheral dopamine (Da) receptor antagonist], or domperidone (Dom, 0.5 mg/kg, a peripheral Da receptor antagonist). In both lean and obese rats, Hal increased tidal volume and decreased respiratory frequency during hyperoxia or normoxia, resulting in an unchanged VE. In contrast, Dom did not affect tidal volume, frequency, or VE during hyperoxia or normoxia. During hypoxia, however, VE significantly increased from 1,132 +/- 136 to 1,348 +/- 98 ml. kg(-1). min(-1) (P < 0.01) after the administration of Dom in obese rats, whereas no change was observed in lean rats. Hal significantly decreased VE during hypoxia compared with control in lean but not obese rats. In both lean and obese rats, Hal decreased VE in response to hypercapnia, whereas Dom had no effect. Our major findings suggest that peripheral chemosensitivity to hypoxia in obese Zucker rats is reduced as a result of an increased dopaminergic receptor modulation in the carotid body.     

Association analysis of gene polymorphism in alcohol metabolizing enzymes with risk for coronary atherosclerosis

The allele and genotype distribution of two alcohol dehydrogenase genes ADH1B (exon 3 polymorphism A/G (47His)), ADH7 (intron 5 polymorphism G/C) and cytochrome P450 2E1 gene (CYP2E1; 5'-flanking region G/C and intron 6 T/A polymorphisms) were examined in Russian (Tomsk, n = 125) healthy population and in coronary atherosclerosis patients (CA, n = 92). The genotype frequencies followed the Hardy-Weinberg equilibrium and the alleles were in linkage equilibrium or gametic equilibrium in the control sample. Only two CYP2E1 gene polymorphisms were in linkage disequilibrium. The frequencies of the derived alleles at ADH1B (*G (+MslI) allele), CYP2E1 (**C2 (+PstI) allele) and CYP2E1 (*C (-Dra I)2 allele) were 8.48 +/- 1.86%; 1.20 +/- 0.69% and 10.00 +/- 1.90%, respectively. The 2ADH7 gene polymorphism showed a high level of heterozygosity; the frequency of the ADH7*C (-Sty I) allele was 44.58 +/- 3.21%. A significantly higher frequency of CYP2E1 (*C2 (+Pst I)) allele has been revealed in the CA group (P = 0.043; OR = 4.23; 95% CI 1.03-20.01). The tendency to significant effect of A1A2 genotype in ADH1B Msl 1 polymorphism was observed for systolic blood pressure in the control group (P = 0.068). The statistically significant two-way interaction effects of ADH7 StyI and CYP2E1 DraI on diastolic blood pressure (P = 0.029) and on the serum high density lipoprotein level (P = 0,042) were also revealed. Association of A1A2 genotype in ADHIB Msl I polymorphism with reduced amount in a serum of a very low density lipoprotein level (P = 0.045) have also been shown. This may result from multifunctional activity of alcohol metabolizing enzymes and their involvement in many metabolic and free radical reactions in the body.     

Crystallization and preliminary X-ray crystallographic studies of Thermus thermophilus HB8 MutM protein involved in repairs of oxidative DNA damage

MutM protein, which removes the oxidatively damaged DNA base product, 8-oxoguanine (GO), has been crystallized by means of a hanging-drop vapor-diffusion procedure using polyethyleneglycol monomethylether 2000 as a precipitant in 2-(cyclohexylamino) ethanesulfonic acid (CHES) buffer, pH 9.8. The diffraction data derived from oscillation photographs indicate that the crystals belong to the monoclinic system and space group P2(1). The crystals have unit-cell dimensions of a = 45.4 A, b = 62.0 A, c = 99.7 A, and beta = 90.8 degrees. Assuming that the asymmetric unit contains two molecules, the Vm value was calculated to be 2.35 A(3).Da(-1). The crystals diffracted X-rays to at least 2.1 A resolution and were suitable for high-resolution X-ray crystal structure determination.     

Preliminary crystallographic analysis of the ATP-hydrolysing domain of the Escherichia coli DNA gyrase B protein.

The 43 kDa N-terminal ATPase domain of the Escherichia coli DNA gyrase B protein has been purified from an over-expressing strain. This protein has been crystallized in two crystal forms, both in the presence of the non-hydrolysable ATP analogue 5'-adenylyl-beta,gamma-imidodiphosphate. The first crystal form is monoclinic P2(1), with cell dimensions a = 76 A, b = 88 A, c = 82 A, beta = 105.5 degrees, and diffracts to at least 2.7 A resolution using synchrotron radiation. Crystal density measurements suggest that there are two molecules in the asymmetric unit (Vm = 3.08 A3/Da). The second crystal form is orthorhombic C222(1), with cell dimensions a = 89.2 A, b = 143.1 A and c = 79.8 A. The crystals diffract to beyond 3 A and are stable for at least 100 hours when exposed to X-rays from a rotating anode source. The asymmetric unit of this crystal form appears to contain one molecule (Vm = 2.96 A3/Da). Data have already been collected to 5 A resolution from native crystals of this second form, and to 6 A resolution from three heavy-atom derivatives. Electron density maps calculated using phases obtained from these derivatives show features consistent with secondary structural elements, and have allowed the molecular boundary to be determined. Higher resolution native and derivative data are being collected.     

Crystallization and preliminary X-ray crystallographic analysis of Mirabilis antiviral protein.

Mirabilis antiviral protein is a single-chain ribosome-inactivating protein purified from the tuberous root of Mirabilis jalapa L. We obtained several forms of crystals of the protein by the hanging drop vapor diffusion method, but most of these crystals were not suitable for X-ray crystallography. After refining the growth conditions, crystals of crystallographic quality were grown in 20-microliters droplets of an equi-volume mixture of 1.5% (w/v) protein solution and a reservoir solution containing 49 to 50% (w/v) ammonium sulfate and 50 mM-ammonium citrate (pH 5.4) at room temperature. Addition of 2 mM-adenine sulfate reduced twinning and "crystal shower". The resulting trigonal crystals diffract beyond 2.5 A resolution using a rotating anode X-ray generator. The space group was determined to be P3(1)21 or P3(2)21 (a = b = 103.9.A, c = 134.6 A, alpha = beta = 90 degrees, gamma = 120 degrees) based on their precession photography of h0l and hk0 zones. There seems to be three monomers in an asymmetric unit for VM = 2.51 A3/Da.     

Spinotrapezius muscle microcirculatory function: effects of surgical exteriorization

Intravital microscopy facilitates insights into muscle microcirculatory structural and functional control, provided that surgical exteriorization does not impact vascular function. We utilized a novel combination of phosphorescence quenching, microvascular oxygen pressure (microvascular PO(2)), and microsphere (blood flow) techniques to evaluate static and dynamic behavior within the exposed intact (I) and exteriorized (EX) rat spinotrapezius muscle. I and EX muscles were studied under control, metabolic blockade with 2,4-dinitrophenol (DNP), and electrically stimulated conditions with 1-Hz contractions, and across switches from 21 to 100% and 10% inspired O(2). Surgical preparation did not alter spinotrapezius muscle blood flow in either I or EX muscle. DNP elevated muscle blood flow approximately 120% (P < 0.05) in both I and EX muscles (P > 0.05 between I and EX). Contractions reduced microvascular PO(2) from 30.4 +/- 4.3 to 21.8 +/- 4.8 mmHg in I muscle and from 33.2 +/- 3.0 to 25.9 +/- 2.8 mmHg in EX muscles with no difference between I and EX. In each O(2) condition, there was no difference (each P > 0.05) in microvascular PO(2) between I and EX muscles (21% O(2): I = 37 +/- 1; EX = 36 +/- 1; 100%: I = 62 +/- 5; EX = 51 +/- 9; 10%: I = 20 +/- 1; EX = 17 +/- 2 mmHg). Similarly, the dynamic behavior of microvascular PO(2) to altered inspired O(2) was unaffected by the EX procedure [half-time (t(1/2)) to 100% O(2): I = 23 +/- 5; EX = 23 +/- 4; t(1/2) to 10%: I = 14 +/- 2; EX = 16 +/- 2 s, both P > 0.05]. These results demonstrate that the spinotrapezius muscle can be EX without significant alteration of microvascular integrity and responsiveness under the conditions assessed.