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V Fülöp

Publications and source records attributed to V Fülöp.

At least 19 recordsLinked to original sources
PubMed2001

Structures of prolyl oligopeptidase substrate/inhibitor complexes. Use of inhibitor binding for titration of the catalytic histidine residue.

Structure determination of the inactive S554A variant of prolyl oligopeptidase complexed with an octapeptide has shown that substrate binding is restricted to the P4-P2' region. In addition, it has revealed a hydrogen bond network of potential catalytic importance not detected in other serine peptidases. This involves a unique intramolecular hydrogen bond between the P1' amide and P2 carbonyl groups and another between the P2' amide and Nepsilon2 of the catalytic histidine 680 residue. It is argued that both hydrogen bonds promote proton transfer from the imidazolium ion to the leaving group. Another complex formed with the product-like inhibitor benzyloxycarbonyl-glycyl-proline, indicating that the carboxyl group of the inhibitor forms a hydrogen bond with the Nepsilon2 of His(680). Because a protonated histidine makes a stronger interaction with the carboxyl group, it offers a possibility of the determination of the real pK(a) of the catalytic histidine residue. This was found to be 6.25, lower than that of the well studied serine proteases. The new titration method gave a single pK(a) for prolyl oligopeptidase, whose reaction exhibited a complex pH dependence for k(cat)/K(m), and indicated that the observed pK(a) values are apparent. The procedure presented may be applicable for other serine peptidases.

Amino Acid Substitution↗
PubMed2001

High anti-paternal cytotoxic T-lymphocyte precursor frequencies in women with unexplained recurrent spontaneous abortions.

A number of cases of unexplained (idiopathic) recurrent spontaneous abortions may be attributable to immunological mechanisms. Several lines of evidence indicate that some immunocompetent effector cell populations play an important role in the pathogenesis of unexplained miscarriages. However a suitable method is lacking for defining an existing immunological background of recurrent spontaneous abortions. We tried to find a useful cellular immunological method, that is suitable for predicting the eventual immunological cause in the case of unexplained recurrent spontaneous abortions. We have examined the anti-paternal cytotoxic T-lymphocyte precursor frequencies by cell-mediated lympholysis and limiting dilution analysis in the peripheral blood of women with recurrent spontaneous abortions in order to reveal the functional role of this cell population in spontaneous abortions. An extremely high partner allo-antigen-specific cytotoxic T-lymphocyte precursor frequency was determined in the case of all those habitual aborters, where no other than an immunological cause could be responsible for the abortions. This phenomenon supports the important role of the T-lymphocytes in this disorder. We suggest that the immunological background of recurrent spontaneous miscarriages might be determined on the basis of a very high cytotoxic T-lymphocyte precursor frequency. This diagnostic test might be useful in selecting patients for immunotherapy.

Abortion, Habitual↗
PubMed2000

Placental site trophoblastic tumor. Clinical and pathological report of two cases.

Placental site trophoblastic tumor (PSTT) is the rarest disease of the gestational trophoblast. Our two cases will be interesting not only because of the rarity of the disease, but because both were recognized before operation. Since the tumor cells are lined up tightly side by side, this disease must be distinguished primarily from tumors of epithelial origin. The authors highlight that the diagnosis should rely on intense hPL-positivity as well as the ultrastructural image of the tumor. In histologically equivocal cases, the determination of hPL, hCG, and MIB-1 immunologic markers can be recommended as routinely performed morphological examinations. Serum hCG monitoring is recommended to follow the evolution of the tumor.

Adult↗
PubMed2000

Catalysis of serine oligopeptidases is controlled by a gating filter mechanism.

Proteases have a variety of strategies for selecting substrates in order to prevent uncontrolled protein degradation. A recent crystal structure determination of prolyl oligopeptidase has suggested a way for substrate selection involving an unclosed seven-bladed beta-propeller domain. We have engineered a disulfide bond between the first and seventh blades of the propeller, which resulted in the loss of enzymatic activity. These results provided direct evidence for a novel strategy of regulation in which oscillating propeller blades act as a gating filter during catalysis, letting small peptide substrates into the active site while excluding large proteins to prevent accidental proteolysis.

Amino Acid Substitution↗
PubMed2000

Crystallization and preliminary X-ray analysis of nitrous oxide reductase from Paracoccus pantotrophus.

Nitrous oxide reductase is a periplasmic respiratory protein with a novel copper catalytic centre; it catalyses the terminal step, reduction of nitrous oxide to nitrogen, of the bacterial denitrification process. Nitrous oxide reductase from Paracoccus pantotrophus has been crystallized by the hanging-drop method. A prerequisite for crystallization was the oxidation of the enzyme with potassium ferricyanide in order to obtain homogenous oxidation states of the copper centres. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 116.4, b = 118.3, c = 267.0 A. Two homodimers, of approximate molecular weight 67 kDa per subunit, probably constitute the asymmetric unit and give a Matthews coefficient, V(m), of 3.4 A(3) Da(-1) and a solvent content of 59% by volume. The crystals diffract X-rays to 3.0 A resolution on an in-house source and are suitable for structure determination.

Crystallization↗
PubMed1999

Beta propellers: structural rigidity and functional diversity.

Recently solved structures and proposed models have helped to reveal the structural characteristics of the beta-propeller fold, as well as the features that contribute to its high rigidity and stability. Possible strategies for identifying beta-propeller proteins in newly characterised sequences are helping to overcome the problems of predicting the beta-propeller fold from amino acid sequences.

Amino Acid Sequence↗
PubMed1998

Prolyl oligopeptidase: an unusual beta-propeller domain regulates proteolysis.

Prolyl oligopeptidase is a large cytosolic enzyme that belongs to a new class of serine peptidases. The enzyme is involved in the maturation and degradation of peptide hormones and neuropeptides, which relate to the induction of amnesia. The 1.4 A resolution crystal structure is presented here. The enzyme contains a peptidase domain with an alpha/beta hydrolase fold, and its catalytic triad (Ser554, His680, Asp641) is covered by the central tunnel of an unusual beta propeller. This domain makes prolyl oligopeptidase an oligopeptidase by excluding large structured peptides from the active site. In this way, the propeller protects larger peptides and proteins from proteolysis in the cytosol. The structure is also obtained with a transition state inhibitor, which may facilitate drug design to treat memory disorders.

Amino Acid Sequence↗
PubMed1997

Haem-ligand switching during catalysis in crystals of a nitrogen-cycle enzyme.

Cytochrome cd1 nitrite reductase catalyses the conversion of nitrite to nitric oxide in the nitrogen cycle. The crystal structure of the oxidized enzyme shows that the d1 haem iron of the active site is ligated by His/Tyr side chains, and the c haem iron is ligated by a His/His ligand pair. Here we show that both haems undergo re-ligation during catalysis. Upon reduction, the tyrosine ligand of the d1 haem is released to allow substrate binding. Concomitantly, a refolding of the cytochrome c domain takes place, resulting in an unexpected change of the c haem iron coordination from His 17/His 69 to Met106/His69. This step is similar to the last steps in the folding of cytochrome c. The changes must affect the redox potential of the haems, and suggest a mechanism by which internal electron transfer is regulated. Structures of reaction intermediates show how nitric oxide is formed and expelled from the active-site iron, as well as how both haems return to their starting coordination. These results show how redox energy can be switched into conformational energy within a haem protein.

Catalysis↗
PubMed1997

N-terminal arm exchange is observed in the 2.15 A crystal structure of oxidized nitrite reductase from Pseudomonas aeruginosa.

BACKGROUND: Nitrite reductase from Pseudomonas aeruginosa (NiR-Pa) is a dimer consisting of two identical 60 kDa subunits, each of which contains one c and one d1 heme group. This enzyme, a soluble component of the electron-transfer chain that uses nitrate as a source of energy, can be induced by the addition of nitrate to the bacterial growth medium. NiR-Pa catalyzes the reduction of nitrite (NO2-) to nitric oxide (NO); in vitro, both cytochrome c551 and azurin are efficient electron donors in this reaction. NiR is a key denitrification enzyme, which controls the rate of the production of toxic nitric oxide (NO) and ultimately regulates the release of NO into the atmosphere. RESULTS: The structure of the orthorhombic form (P2(1)2(1)2) of oxidized NiR-Pa was solved at 2.15 A resolution, using molecular replacement with the coordinates of the NiR from Thiosphaera pantotropha (NiR-Tp) as the starting model. Although the d1-heme domains are almost identical in both enzyme structures, the c domain of NiR-Pa is more like the classical class I cytochrome-c fold because it has His51 and Met88 as heme ligands, instead of His17 and His69 present in NiR-Tp. In addition, the methionine-bearing loop, which was displaced by His17 of the NiR-Tp N-terminal segment, is back to normal in our structure. The N-terminal residues (5/6-30) of NiR-Pa and NiR-Tp have little sequence identity. In Nir-Pa, this N-terminal segment of one monomer crosses the dimer interface and wraps itself around the other monomer. Tyr10 of this segment is hydrogen bonded to an hydroxide ion--the sixth ligand of the d1-heme Fe, whereas the equivalent residue in NiR-Tp, Tyr25, is directly bound to the Fe. CONCLUSIONS: Two ligands of hemes c and d1 differ between the two known NiR structures, which accounts for the fact that they have quite different spectroscopic and kinetic features. The unexpected domain-crossing by the N-terminal segment of NiR-Pa is comparable to that of 'domain swapping' or 'arm exchange' previously observed in other systems and may explain the observed cooperativity between monomers of dimeric NiR-Pa. In spite of having similar sequence and fold, the different kinetic behaviour and the spectral features of NiR-Pa and NiR-Tp are tuned by the N-terminal stretch of residues. A further example of this may come from another NiR, from Pseudomonas stutzeri, which has an N terminus very different from that of the two above mentioned NiRs.

Amino Acid Sequence↗
PubMed1997

Cytochrome cd1 structure: unusual haem environments in a nitrite reductase and analysis of factors contributing to beta-propeller folds.

The central tunnel of the eight-bladed beta-propeller domain of cytochrome cd1 (nitrite reductase) is seen, from a 1.28 A resolution structure, to contain hydrogen donors and acceptors that are satisfied by interaction either with water or the d1 haem. The d1 haem, although bound by an extensive network of hydrogen bonds, is not distorted in its binding pocket and is confirmed to have exactly the dioxoisobacteriochlorin structure proposed from chemical studies. A biological rationale is advanced for the undistorted structure of the d1 haem and the large number of hydrogen bonds it makes. The beta-propeller domain can be closely superimposed on that of methanol dehydrogenase despite the enzymes sharing no common sequence motifs and using a different set of interactions to "Velcro" close the propeller. The sequence and likely structural relationships between cytochrome cd1 or methanol dehydrogenase and other predicted eight-bladed beta-propeller domains in proteins, such as the pyrolloquinoline quinone-dependent alcohol dehydrogenase, are discussed and compared with other propeller proteins. From sequencing the nirS gene of Thiosphaera pantotropha, it is established that the amino acid sequence deduced previously in part from X-ray diffraction data at lower resolution was largely correct, as was the proposal that eight N-terminal amino acid residues were not seen in the structure. The unusual haem iron environments in both the c-type cytochrome domain, with His/His coordination, and the d1-type cytochrome domain with Tyr/His coordination are related to the functions of the redox centres.

Amino Acid Sequence↗
PubMed1995

Crystal structure of the di-haem cytochrome c peroxidase from Pseudomonas aeruginosa.

BACKGROUND: Cytochrome c peroxidase from Pseudomonas aeruginosa (PsCCP) represents a new class of peroxidases which work without the need to create a semi-stable free radical for catalysis. The enzyme is located in the bacterial periplasm where its likely function is to provide protection against toxic peroxides. The soluble 323-residue single polypeptide chain contains two covalent c-type haems with very different properties: one of them is a low-potential (-330 mV) centre where hydrogen peroxide is reduced (the peroxidatic site); the other is a high-potential (+320 mV) centre which feeds electrons to the peroxidatic site from soluble electron-shuttle proteins such as cytochrome c and azurin. RESULTS: The crystal structure of the oxidized form of PsCCP has been determined to 2.4 A resolution by multiple isomorphous replacement, and refined to an R-factor of 19.2%. PsCCP is organized into two domains, both of them containing a covalent c-haem in a structure reminiscent of class 1 cytochromes c. The domains are related by a quasi-twofold axis. The domain interface holds a newly discovered calcium-binding site with an unusual set of ligands. CONCLUSIONS: The likely function of the calcium site is to maintain the structural integrity of the enzyme and/or to modulate electron transfer between the two haem domains. The low-potential haem has two histidine axial ligands (His55 and His71) and the high-potential haem is ligated by His201 and Met275. There are no polar residues at the peroxidatic site in the inactive oxidized enzyme. The structure suggests that, in the half-reduced functional form of the enzyme, the low-potential haem has to shed His71 in order to make the enzyme catalytically competent. This process is likely to trigger a reorganization of the active site, and may introduce a new residues into the haem pocket.

Amino Acid Sequence↗
PubMed1995

Crystal structure of isopenicillin N synthase is the first from a new structural family of enzymes.

Penicillin antibiotics are all produced from fermentation-derived penicillins because their chemical synthesis is not commercially viable. The key step in penicillin biosynthesis, in which both the beta-lactam and thiazolidine rings of the nucleus are created, is mediated by isopenicillin N synthase (IPNS), which binds ferrous iron and uses dioxygen as a cosubstrate. In a unique enzymatic step, with no chemical precedent, IPNS catalyses the transfer of four hydrogen atoms from its tripeptide substrate to dioxygen forming, in a single reaction, the complete bicyclic nucleus of the penicillins. We now report the structure of IPNS complexed with manganese, which reveals the active site is unusually buried within a 'jelly-roll' motif and lined by hydrophobic residues, and suggest how this structure permits the process of penicillin formation. Sequence analyses indicate IPNS, 1-aminocyclopropane-1-carboxylic acid oxidase and many of the 2-oxo-acid-dependent oxygenases contain a conserved jelly-roll motif, forming a new structural family of enzymes.

Acremonium↗
PubMed1995

The anatomy of a bifunctional enzyme: structural basis for reduction of oxygen to water and synthesis of nitric oxide by cytochrome cd1.

Cytochrome cd1-nitrite reductase is a bifunctional enzyme that catalyzes the one-electron reduction of nitrite to nitric oxide and the four-electron reduction of oxygen to water. The 1.55 A crystal structure of the dimeric enzyme from Thiosphaera pantotropha is reported here. The protein was sequenced from the X-ray structure. Each subunit contains a covalent c heme with two axial His ligands (His-17, His-69) and a unique noncovalent d1 heme ligated by Tyr-25 and His-200. The d1 heme is the mononuclear iron center where both oxygen and nitrite reduction take place. The two types of heme are located in separate domains whose arrangement suggests a mechanism requiring domain movement during catalysis.

Amino Acid Sequence↗
PubMed1995

Pseudospecific docking surfaces on electron transfer proteins as illustrated by pseudoazurin, cytochrome c550 and cytochrome cd1 nitrite reductase.

The structure of pseudoazurin from Thiosphaera pantotropha has been determined and compared to structures of both soluble and membrane-bound periplasmic redox proteins. The results show a matching set of unipolar, but promiscuous, docking motifs based on a positive hydrophobic surface patch on the electron shuttle proteins pseudoazurin and cytochrome c550 and a negative hydrophobic patch on the surface of their known redox partners. The observed electrostatic handedness is argued to be associated with the charge-asymmetry of the membrane-bound components of the redox chain due to von Heijne's 'positives-inside' principle. We propose a 'positives-in-between' rule for electron shuttle proteins, and expect a negative hydrophobic patch to be present on both the highest and lowest redox potential species in a series of electron carriers.

Azurin↗
PubMed1995

The polymorphic human TLX-B/CD46/MCP system and its implications in transplantation and reproduction.

TLX antigens have been found on most peripheral blood cells, trophoblasts, seminal vesicle cells and sperms. These antigens seem to be associated with the membrane cofactor protein (MCP) and the CD46 antigen. Alloantibodies to TLX antigens with Fc tau RII-blocking features were obtained by transfusion of leucocytes or platelets. Preliminary population studies revealed that alloantibodies to TLX/CD46/MCP recognize four overlapping specificities. The terminology TLX-B was introduced with specificities TLX-B1, B2, B3, B4 and frequencies obtained in the population were: 38%, 46%, 42% and 26%, respectively. Family studies showed an independent segregation of the TLX and HLA alleles. At the cellular protein on trophoblast, the alloantibody detected a glycoprotein of 66-67 kDa molecular mass, which may correspond to the alpha chain of the TLX/CD46/MCP isotypes. A direct association of the alloantibody with Fc tau RII could be excluded thus its FcR blocking feature is probably based on an indirect functional effect. After transfusion and in pregnancy the induction of TLX alloantibody production depended on the mismatching in the TLX/CD46/MCP phenotypes. Probable associations were revealed in the case of recurrent habitual abortion between the lack of Fc tau R blocking antibody production and the matched TLX specificities of the couples. After transfusion, TLX alloantibody production with Fc tau R and MLR blocking function was induced only when the recipient was lacking the TLX specificities expressed on the donor cells. Suppression of MLR was found only when TLX specificity in sera corresponded to the TLX specificity of the effector cell. The immunopathological importance of these findings in transplantation and reproductive medicine has yet to be clarified.

Abortion, Habitual↗
PubMed1994

[Detection of gamma-interferon mRNA in JEG-3 choriocarcinoma cell line by means of polymerase chain reaction].

To investigate the pathogenesis of choriocarcinoma the authors employed a newly developed gene amplification method by reverse polymerase chain reaction for the detection of gamma-interferon messenger RNA in JEG-3 choriocarcinoma cell line. Polymerase chain reaction products were analysed by agarose gel electrophoresis. Using 1 Kb DNA ladder as a marker, 84 base-pair fragment was selectively amplified correlating with published gamma-interferon gene fragment length. Because cDNA contains a virtually complete copy of the mRNA this method provides an evidence for the expression of gamma-interferon gene in JEG-3 choriocarcinoma cell line. Based on these results a potential autocrine mechanism may be present in JEG-3 choriocarcinoma cell line.

Choriocarcinoma↗
PubMed1994

Laue diffraction study on the structure of cytochrome c peroxidase compound I.

BACKGROUND: Cytochrome c peroxidase from yeast is a soluble haem-containing protein found in the mitochondrial electron transport chain where it probably protects against toxic peroxides. The aim of this study was to obtain a reliable structure for the doubly oxidized transient intermediate (termed compound I) in the reaction of cytochrome c peroxidase with hydrogen peroxide. This intermediate contains a semistable free radical on Trp191, and an oxyferryl haem group. RESULTS: Compound I was produced in crystals of yeast cytochrome c peroxidase by reacting the crystalline enzyme with hydrogen peroxide in a flow cell. The reaction was monitored by microspectrophotometry and Laue crystallography in separate experiments. A nearly complete conversion to compound I was achieved within two minutes of the addition of hydrogen peroxide, and the concentration of the intermediate remained at similar levels for an additional half an hour. The structure of the intermediate was determined by Laue diffraction. The refined Laue structure for compound I shows clear structural changes at the peroxide-binding site but no significant changes at the radical site. The photographs were processed with a new software package (LEAP), overcoming many of the former problems encountered in extracting structural information from Laue exposures. CONCLUSIONS: The geometry of the haem environment in this protein allows structural changes to be extremely small, similar in magnitude to those observed for the Fe2+/Fe3+ transition in cytochrome c. The results suggest that these molecules have evolved to transfer electrons with a minimal need for structural adjustment.

Amino Acid Sequence↗