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O Bârzu

Publications and source records attributed to O Bârzu.

At least 19 recordsLinked to original sources
PubMed2001

X-ray structure of TMP kinase from Mycobacterium tuberculosis complexed with TMP at 1.95 A resolution.

The X-ray structure of Mycobacterium tuberculosis TMP kinase at 1.95 A resolution is described as a binary complex with its natural substrate TMP. Its main features involve: (i) a clear magnesium-binding site; (ii) an alpha-helical conformation for the so-called LID region; and (iii) a high density of positive charges in the active site. There is a network of interactions involving highly conserved side-chains of the protein, the magnesium ion, a sulphate ion mimicking the beta phosphate group of ATP and the TMP molecule itself. All these interactions conspire in stabilizing what appears to be the closed form of the enzyme. A complete multialignment of all (32) known sequences of TMP kinases is presented. Subtle differences in the TMP binding site were noted, as compared to the Escherichia coli, yeast and human enzyme structures, which have been reported recently. These differences could be used to design specific inhibitors of this essential enzyme of nucleotide metabolism. Two cases of compensatory mutations were detected in the TMP binding site of eukaryotic and prokaryotic enzymes. In addition, an intriguing high value of the electric field is reported in the vicinity of the phosphate group of TMP and the putative binding site of the gamma phosphate group of ATP.

Adenosine Triphosphate↗
PubMed2000

Substrate-induced fit of the ATP binding site of cytidine monophosphate kinase from Escherichia coli: time-resolved fluorescence of 3'-anthraniloyl-2'-deoxy-ADP and molecular modeling.

The conformation and dynamics of the ATP binding site of cytidine monophosphate kinase from Escherichia coli (CMPK(coli)), which catalyzes specifically the phosphate exchange between ATP and CMP, was studied using the fluorescence properties of 3'-anthraniloyl-2'-deoxy-ADP, a specific ligand of the enzyme. The spectroscopic properties of the bound fluorescent nucleotide change strongly with respect to those in aqueous solution. These changes (red shift of the absorption and excitation spectra, large increase of the excited state lifetime) are compared to those observed in different solvents. These data, as well as acrylamide quenching experiments, suggest that the anthraniloyl moiety is protected from the aqueous solvent upon binding to the ATP binding site, irrespective of the presence of CMP or CDP. The protein-bound ADP analogue exhibits a restricted fast subnanosecond rotational motion, completely blocked by CMP binding. The energy-minimized models of CMPK(coli) complexed with 3'-anthraniloyl-2'-deoxy-ADP using the crystal structures of the ligand-free protein and of its complex with CDP (PDB codes and, respectively) were compared to the crystal structure of UMP/CMP kinase from Dictyostelium discoideum complexed with substrates (PDB code ). The key residues for ATP/ADP binding to CMPK(coli) were identified as R157 and I209, their side chains sandwiching the adenine ring. Moreover, the residues involved in the fixation of the phosphate groups are conserved in both proteins. In the model, the accessibility of the fluorescent ring to the solvent should be substantial if the LID conformation remained unchanged, by contrast to the fluorescence data. These results provide the first experimental arguments about an ATP-mediated induced-fit of the LID in CMPK(coli) modulated by CMP, leading to a closed conformation of the active site, protected from water.

Adenosine Triphosphate↗
PubMed2000

Genetic and biochemical characterization of Salmonella enterica serovar typhi deoxyribokinase.

We identified in the genome of Salmonella enterica serovar Typhi the gene encoding deoxyribokinase, deoK. Two other genes, vicinal to deoK, were determined to encode the putative deoxyribose transporter (deoP) and a repressor protein (deoQ). This locus, located between the uhpA and ilvN genes, is absent in Escherichia coli. The deoK gene inserted on a plasmid provides a selectable marker in E. coli for growth on deoxyribose-containing medium. Deoxyribokinase is a 306-amino-acid protein which exhibits about 35% identity with ribokinase from serovar Typhi, S. enterica serovar Typhimurium, or E. coli. The catalytic properties of the recombinant deoxyribokinase overproduced in E. coli correspond to those previously described for the enzyme isolated from serovar Typhimurium. From a sequence comparison between serovar Typhi deoxyribokinase and E. coli ribokinase, whose crystal structure was recently solved, we deduced that a key residue differentiating ribose and deoxyribose is Met10, which in ribokinase is replaced by Asn14. Replacement by site-directed mutagenesis of Met10 with Asn decreased the V(max) of deoxyribokinase by a factor of 2.5 and increased the K(m) for deoxyribose by a factor of 70, compared to the parent enzyme.

Amino Acid Sequence↗
PubMed1999

Decreased capacity of recombinant 45/47-kDa molecules (Apa) of Mycobacterium tuberculosis to stimulate T lymphocyte responses related to changes in their mannosylation pattern.

The Apa molecules secreted by Mycobacterium tuberculosis, Mycobacterium bovis, or BCG have been identified as major immunodominant antigens. Mass spectrometry analysis indicated similar mannosylation, a complete pattern from 1 up to 9 hexose residues/mole of protein, of the native species from the 3 reference strains. The recombinant antigen expressed in M. smegmatis revealed a different mannosylation pattern: species containing 7 to 9 sugar residues/mole of protein were in the highest proportion, whereas species bearing a low number of sugar residues were almost absent. The 45/47-kDa recombinant antigen expressed in E. coli was devoid of sugar residues. The proteins purified from M. tuberculosis, M. bovis, or BCG have a high capacity to elicit in vivo potent delayed-type hypersensitivity (DTH) reactions and to stimulate in vitro sensitized T lymphocytes of guinea pigs immunized with living BCG. The recombinant Apa expressed in Mycobacterium smegmatis was 4-fold less potent in vivo in the DTH assay and 10-fold less active in vitro to stimulate sensitized T lymphocytes than the native proteins. The recombinant protein expressed in Escherichia coli was nearly unable to elicit DTH reactions in vivo or to stimulate T lymphocytes in vitro. Thus the observed biological effects were related to the extent of glycosylation of the antigen.

Amino Acid Sequence↗
PubMed1999

The highly similar TMP kinases of Yersinia pestis and Escherichia coli differ markedly in their AZTMP phosphorylating activity.

Thymidine monophosphate (TMP) kinases are key enzymes in nucleotide synthesis for all living organisms. Although eukaryotic and viral TMP kinases have been studied extensively, little is known about their bacterial counterparts. To characterize the TMP kinase of Yersinia pestis, a chromosomal region encompassing its gene (tmk) was cloned and sequenced; a high degree of conservation with the corresponding region of Escherichia coli was found. The Y. pestis tmk gene was overexpressed in E. coli, where the enzyme represented over 20% of total soluble proteins. The CD spectrum of the purified TMP kinase from Y. pestis was characteristic for proteins rich in alpha-helical structures. Its thermodynamic stability was significantly lower than that of E. coli TMP kinase. However, the most striking difference between the two enzymes was related to their ability to phosphorylate 3'-deoxy-3'-azidothymidine monophosphate (AZTMP). Although the enzymes of both species had comparable Km values for this analogue, they differed significantly in their Vmax for AZTMP. Whereas E. coli used AZTMP as a relatively good substrate, the Y. pestis enzyme had a Vmax 100 times lower with AZTMP than with TMP. This fact explains why AZT, a potent bactericidal agent against E. coli, is only moderately active on Y. enterocolitica. Sequence comparisons between E. coli and Y. pestis TMP kinases along with the three-dimensional structure of the E. coli enzyme suggest that segments lying outside the main regions involved in nucleotide binding and catalysis are responsible for the different rates of AZTMP phosphorylation.

Amino Acid Sequence↗
PubMed1999

Multinuclear magnetic resonance studies of Escherichia coli adenylate kinase in free and bound forms. Resonance assignment, secondary structure and ligand binding.

The crystal structure of Escherichia coli adenylate kinase (AKe) revealed three main components: a CORE domain, composed of a five-stranded parallel beta-sheet surrounded by alpha-helices, and two peripheral domains involved in covering the ATP in the active site (LID) and binding of the AMP (NMPbind). We initiated a long-term NMR study aiming to characterize the solution structure, binding mechanism and internal dynamics of the various domains. Using single (15N) and double-labeled (13C and 15N) samples and double- and triple-resonance NMR experiments we assigned 97% of the 1H, 13C and 15N backbone resonances, and proton and 13Cbeta resonances for more than 40% of the side chains in the free protein. Analysis of a 15N-labeled enzyme in complex with the bi-substrate analogue [P1,P5-bis(5'-adenosine)-pentaphosphate] (Ap5A) resulted in the assignment of 90% of the backbone 1H and 15N resonances and 42% of the side chain resonances. Based on short-range NOEs and 1H and 13C secondary chemical shifts, we identified the elements of secondary structure and the topology of the beta-strands in the unliganded form. The alpha-helices and the beta-strands of the parallel beta-sheet in solution have the same limits (+/- 1 residue) as those observed in the crystal. The first helix (alpha1) appears to have a frayed N-terminal side. Significant differences relative to the crystal were noticed in the LID domain, which in solution exhibits four antiparallel beta-strands. The secondary structure of the nucleoside-bound form, as deduced from intramolecular NOEs and the 1Halpha chemical shifts, is similar to that of the free enzyme. The largest chemical shift differences allowed us to map the regions of protein-ligand contacts. 1H/2H exchange experiments performed on free and Ap5A-bound enzymes showed a general decrease of the structural flexibility in the complex which is accompanied by a local increased flexibility on the N-side of the parallel beta-sheet.

Adenylate Kinase↗
PubMed1999

Immunochemical analysis of UMP kinase from Escherichia coli.

Mono- and polyclonal antibodies directed against UMP kinase from Escherichia coli were tested with the intact protein or with fragments obtained by deletion mutagenesis. As detected in enzyme-linked immunosorbent assay tests, the carboxy-terminal quarter of UMP kinase is immunodominant. Polyclonal antibodies inhibited the enzyme activity with partial or total loss of allosteric effects exerted by UTP and GTP, respectively. These data indicate that the UTP and GTP binding sites in UMP kinase are only partially overlapping. One monoclonal antibody (44-2) recognized a linear epitope in UMP kinase between residues 171 and 180. A single substitution (D174N) in this segment of the enzyme abolished its interaction with the monoclonal antibody (44-2). Polyclonal antisera were used to identify UMP kinase in the bacterial proteome. The enzyme appears as a single spot on two-dimensional electrophoresis at a pI of 7.24 and an apparent molecular mass of 26 kDa. Immunogold labeling of UMP kinase in whole E. coli cells shows a localization of the protein near the bacterial membranes. Because the protein does not contain sequences usually required for compartmentalization, the aggregation properties of UMP kinase observed in vitro might play a role in this phenomenon. The specific localization of UMP kinase might also be related to its putative role in cell division.

Animals↗
PubMed1998

Structures of escherichia coli CMP kinase alone and in complex with CDP: a new fold of the nucleoside monophosphate binding domain and insights into cytosine nucleotide specificity.

BACKGROUND: . Nucleoside monophosphate kinases (NMP kinases) catalyze the reversible transfer of a phosphoryl group from a nucleoside triphosphate to a nucleoside monophosphate. Among them, cytidine monophosphate kinase from Escherichia coli has a striking particularity: it is specific for CMP, whereas in eukaryotes a unique UMP/CMP kinase phosphorylates both CMP and UMP with similar efficiency. RESULTS: . The crystal structure of the CMP kinase apoenzyme from E. coli was solved by single isomorphous replacement and refined at 1.75 A resolution. The structure of the enzyme in complex with CDP was determined at 2.0 A resolution. Like other NMP kinases, the protein contains a central parallel beta sheet, the strands of which are connected by alpha helices. The enzyme differs from other NMP kinases in the presence of a 40-residue insert situated in the NMP-binding (NMPbind) domain. This insert contains two domains: one comprising a three-stranded antiparallel beta sheet, the other comprising two alpha helices. CONCLUSIONS: . Two features of the CMP kinase from E. coli have no equivalent in other NMP kinases of known structure. Firstly, the large NMPbind insert undergoes a CDP-induced rearrangement: its beta-sheet domain moves away from the substrate, whereas its helical domain comes closer to it in a motion likely to improve the protection of the active site. Secondly, residues involved in CDP recognition are conserved in CMP kinases and have no counterpart in other NMP kinases. The structures presented here are the first of a new family of NMP kinases specific for CMP.

Amino Acid Sequence↗
PubMed1998

Mutational analysis of UMP kinase from Escherichia coli.

UMP kinase from Escherichia coli is one of the four regulatory enzymes involved in the de novo biosynthetic pathway of pyrimidine nucleotides. This homohexamer, with no counterpart in eukarya, might serve as a target for new antibacterial drugs. Although the bacterial enzyme does not show sequence similarity with any other known nucleoside monophosphate kinase, two segments between amino acids 35 to 78 and 145 to 194 exhibit 28% identity with phosphoglycerate kinase and 30% identity with aspartokinase, respectively. Based on these similarities, a number of residues of E. coli UMP kinase were selected for site-directed mutagenesis experiments. Biochemical, kinetic, and spectroscopic analysis of the modified proteins identified residues essential for catalysis (Asp146), binding of UMP (Asp174), and interaction with the allosteric effectors, GTP and UTP (Arg62 and Asp77).

Amino Acid Sequence↗
PubMed1997

A new non-heme iron environment in Paracoccus denitrificans adenylate kinase studied by electron paramagnetic resonance and electron spin echo envelope modulation spectroscopy.

Adenylate kinase from the Gram-negative bacterium Paracoccus denitrificans (AKden) has structural features highly similar to those of the enzyme from Gram-positive organisms. Atomic absorption spectroscopy of the recombinant protein, which is a dimer, revealed the presence of two metals, zinc and iron, each binding most probably to one monomer. Under oxidizing conditions, the electron paramagnetic resonance (EPR) spectrum of AKden at 4.2 K consists of features at g = 9.23, 4.34, 4.21, and 3.68. These features are absent in the ascorbate-reduced protein and are characteristic of a S = 5/2 spin system in a rhombic environment with E/D = 0.24 and are assigned to a non-heme Fe3+ (S = 5/2) center. The zero-field splitting parameter D (D = 1.4 +/- 0.2 cm-1) was estimated from the temperature dependence of the EPR spectra. These EPR characteristic as well as the difference absorption spectrum (oxidized minus reduced) of AKden are similar to those reported for the non-heme iron protein rubredoxin. Nevertheless, the redox potential of the Fe2+/Fe3+ couple in AKden was measured at +230 +/- 30 mV, which is more positive than the redox potential of the non-heme iron in rubredoxin. Binding of cyanide converts the iron from the high-spin (S = 5/2) to the low-spin (S = 1/2) spin state. The EPR spectrum of the non-heme Fe3+(S = 1/2) in the presence of cyanide has g values of 2.45, 2.18, and 1.92 and spin-Hamiltonian parameters R/lambda = 7. 4 and R/mu = 0.56. The conversion of the non-heme iron to the low-spin (S = 1/2) state allowed the study of its local environment by electron spin echo envelope modulation spectroscopy (ESEEM). The ESEEM data revealed the existence of 14N or 15N nuclei coupled to the low-spin iron after addition of KC14N or KC15N respectively. This demonstrated that iron in AKden has at least one labile coordination position that can be easily occupied by cyanide. Other possible magnetic interactions with nitrogen(s) from the protein are discussed.

Adenylate Kinase↗
PubMed1997

Borrelia burgdorferi uridine kinase: an enzyme of the pyrimidine salvage pathway for endogenous use of nucleotides.

The 621 bp udk gene encoding Borrelia burgdorferi potential uridine kinase, involved in the pyrimidine salvage pathway, was cloned and sequenced. The B burgdorferi protein has a molecular mass of 24 kDa in sodium dodecyl sulfate-polyacrylamide gel. The N-terminal sequence of the protein, Ala-Lys-Ile-Ile, is identical to that predicted but lacks N-terminal methionine. udk is located at around 15 kb from the left telomere and forms an operon with an upstream ORF. A likely hypothesis for the role of the pyrimidine salvage pathway is the sole use of endogenous nucleotides for Borrelia.

Amino Acid Sequence↗
PubMed1997

Structural and catalytic properties of CMP kinase from Bacillus subtilis: a comparative analysis with the homologous enzyme from Escherichia coli.

CMP kinases from Bacillus subtilis and from Escherichia coli are encoded by the cmk gene (formerly known as jofC in B. subtilis and as mssA in E. coli). Similar in their primary structure (43% identity and 67% similarity in amino acid sequence), the two proteins exhibit significant differences in nucleotide binding and catalysis. ATP, dATP, and GTP are equally effective as phosphate donors with E. coli CMP kinase whereas GTP is a poor substrate with B. subtilis CMP kinase. While CMP and dCMP are the best phosphate acceptors of both CMP kinases, the specific activity with these substrates and ATP as donor are 7- to 10-fold higher in the E. coli enzyme; the relative Vm values with UMP and CMP are 0.1 for the B. subtilis CMP kinase and 0.01 for the E. coli enzyme. CMP increased the affinity of E. coli CMP kinase for ATP or for the fluorescent analog 3'-anthraniloyl dATP by one order of magnitude but had no effect on the B. subtilis enzyme. The differences in the catalytic properties of B. subtilis and E. coli CMP kinases might be reflected in the structure of the two proteins as inferred from infrared spectroscopy. Whereas the spectrum of B. subtilis CMP kinase is dominated by a band at 1633 cm-1 (representing beta type structures), the spectrum of the E. coli enzyme is dominated by two bands at 1653 and 1642 cm-1 associated with alpha-helical and unordered structures, respectively. CMP induced similar spectral changes in both proteins with a rearrangement of some of the beta-structures. ATP increases the denaturation temperature of B. subtilis CMP kinase by 9.3 degrees C, whereas in the case of the E. coli enzyme, binding of ATP has only a minor effect.

Amino Acid Sequence↗
PubMed1997

Characterization of metal and nucleotide liganded forms of adenylate kinase by electrospray ionization mass spectrometry.

Complexes of adenylate kinase from Escherichia coli, Bacillus subtilis, and Bacillus stearothermophilus with the bisubstrate nucleotide analog P1,P5-di(adenosine 5')-pentaphosphate and with metal ions (Zn2+ and/or Mg2+) were analyzed by electrospray ionization mass spectrometry. P1,P5-di(adenosine 5')-pentaphosphate. adenylate kinase complex was detected in the positive mode at pH as low as 3.8. Binding of nucleotide to adenylate kinase stabilizes the overall structure of the protein and preserves the Zn2+ chelated form of the enzyme from the gram-positive organisms. In this way, it is possible in a single mass spectrometry experiment to screen metal-chelating adenylate kinases, without use of radioactively labeled compounds. Binding of Mg2+ to enzyme via P1,P5-di(adenosine 5')-pentaphosphate was also demonstrated by mass spectrometry. Although no amino acid side chain in adenylate kinase is supposed to interact with Mg2+, Asp93 in porcine muscle cytosolic enzyme, equivalent to Asp84 in the E. coli adenylate kinase, was proposed to stabilize the nucleotide.Mg2+ complex via water molecules.

Adenylate Kinase↗
PubMed1996

Structural properties of UMP-kinase from Escherichia coli: modulation of protein solubility by pH and UTP.

UMP-kinase from Escherichia coli, unlike the analogous enzyme from eukaryotic organisms, is an oligomeric protein subjected to complex regulatory mechanisms in which UTP and GTP act as allosteric effectors. While the enzyme has an unusually low solubility at neutral pH (< or = 0.1 mg of protein/ mL), its solubility increases markedly above pH 8 and below pH 4. Furthermore, the solubility of the bacterial UMP-kinase at neutral pH is greatly enhanced in the presence of Mg-free UTP. Thermal denaturation experiments have demonstrated that UTP also increases the stability of the protein. Fourier-transform infrared spectroscopy and circular dichroism show that the secondary structure of the protein is the same at neutral and at alkaline pH. These data indicate that variations in enzyme solubility must be related to subtle changes in the tertiary and/or quaternary structure which modulate the exposure of hydrophobic surfaces in the protein molecule. A variant of UMP-kinase, obtained by site-directed mutagenesis (Asp159Asn), which is similar to the wild-type enzyme in its stability and kinetic properties, has a much increased water solubility (> 5 mg protein/mL) even at neutral pH. This suggests that salt bridges may be involved in the equilibrium between the soluble and aggregated forms of the wild-type enzyme, and that conformational changes induced upon binding of UTP increase the protein solubility by disrupting these salt bridges.

Allosteric Regulation↗