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J Hajdu

Publications and source records attributed to J Hajdu.

At least 55 records · Page 3Linked to original sources
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

Laue diffraction studies of human rhinovirus 14 and canine parvovirus.

Laue diffraction data have been collected from monoclinic crystals of canine parvovirus (CPV), and from cubic crystals of human rhinovirus 14 (HRV14) with and without bound antiviral compounds. In optimal conditions one or two images of HRV14 were sufficient to calculate interpretable electron-density maps of the virus complexes at 3.5 A resolution. The crystals of CPV were of lower symmetry and were more easily damaged by radiation, making it difficult to accumulate a significant amount of useful data. Results on HRV14 are compared in studies on four antiviral compounds where data were collected using both monochromatic and Laue diffraction. Two Laue diffraction images of HRV14 with a point mutation were sufficient to determine the change from a leucine to a valine in VP2.

Journal Article↗
PubMed1995

[Quality of life of post-colostomy patients].

The psychosomatic care of patients after surgical treatment must comprise the analysis of quality of life with colostomy. Five different parameters were assessed of 100 colostomy patients. Sixty-one percent of our patients had skin irritation problems. Twenty-eight patients applied regular irrigation. Seventy-two percent of these had daily motions, 20% had bidaily, the remaining 8% had irregular bowel movements. Seventeen percent of the nonirrigating patients observed very irritating fecal discharge around the stoma. Embarrassing noises concomittant with bowel movements were observed by 42% and 45% struggled with bad odors. Irrigating patients had hardly any of these problems. For 65% of the patients the idea of having a colostoma meant the greatest psychological burden before the operation. The stress situation culminated in the immediate postoperative period in 10%. Forty percent of those asked were seriously worried about the reaction of their social environment. In 65% a significant decrease of social relations could be observed. Sixteen percent reported an increased social activity after colostomy had been performed. Sixty percent admitted considering the option of suicide at least once. Thirty-five percent revealed to have any kind of sexual problems after colostomy. Eight of our 100 colostomy patients experienced serious adverse reactions from their family members. In eighty percent of the cases family members showed great sympathy although they acted naively. The quality of life of colostomy patients may be best taken care of by qualified stomatherapists, out-patient proctology departments and the ileo-colostomy movement (ILCO).

Adult↗
PubMed1995

Refined 3.2 A structure of glycosomal holo glyceraldehyde phosphate dehydrogenase from Trypanosoma brucei brucei.

The three-dimensional crystal structure of the enzyme glyceraldehyde phosphate dehydrogenase from the kinetoplastid Trypanosoma brucei brucei has been determined at 3.2 A resolution from a 37% complete data set collected using the Laue method. The crystals used in the structure determination contain one and a half tetrameric enzyme molecules in the asymmetric unit, i.e. six identical subunits. Initial phasing was carried out by the method of molecular replacement using the refined coordinates of holo glyceraldehyde phosphate dehydrogenase from Bacillus stearothermophilus as a search model. The initial electron-density distribution, obtained from the molecular-replacement solution, was greatly improved by a procedure consisting of 36 cycles of iterative non-crystallographic density averaging. During the averaging procedure, the missing reflections (63% of the data) were gradually introduced as map-inversion structure factors. At completion of the procedure, the R-factor between averaged map-inversion amplitudes and observed structure-factor amplitudes was 19.0% for all data between 7.0 and 3.2 A resolution, and that between the map-inversion amplitudes and later recorded structure-factor amplitudes was 41.9%. After model building into the resulting averaged electron-density map, refinement by molecular-dynamics procedures with X-PLOR provided the current model, which has an R-factor of 17.6% for 34 835 reflections between 7.0 and 3.2 A resolution. The refined model, comprising 2735 protein atoms plus one NAD(+) molecule and two sulfate ions per subunit, has r.m.s. deviations from ideality of 0.02 A for bond lengths and 3.6 degrees for bond angles. All subunits, located either within the tetrameric molecule or within the half tetramer present in the asymmetric unit, are related to each other by almost exact twofold symmetry. The overall structure of the glycosomal glyceraldehyde phosphate dehydrogenase subunit and its quaternary arrangement in the tetrameric molecule are similar to that of the enzyme of lobster and Bacillus stearothermophilus (with r.m.s. differences between equivalent Calpha positions of 0.71 and 0.64 A, respectively). The main differences between the structures is the presence of three insertions, plus the substitution of a beta-strand by a short alpha-helix, both occurring at the surface of the glycosomal enzyme subunit.

Journal Article↗
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

Crystallization and preliminary X-ray diffraction studies on recombinant isopenicillin N synthase from Aspergillus nidulans.

Recombinant Aspergillus nidulans isopenicillin N synthase was purified from an Escherichia coli expression system. The apoenzyme in the presence of saturating concentrations of MnCl2 could be crystallized by either macro- or microseeding, using the hanging drop vapor diffusion technique with polyethylene glycol 8000 as precipitant. The crystals (0.5-1.0 mm overall dimensions) diffract X-rays to at least 2.0 A resolution at synchrotrons and belong to space group P212121 with unit cell dimensions of a = 59.2 A, b = 127.0 A, and c = 139.6 A. The asymmetric unit contains one dimer, and the solvent content of the crystals is 60%. The crystals are radiation sensitive.

Aspergillus nidulans↗
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↗
PubMed1994

Crystallization and preliminary X-ray diffraction studies on a recombinant isopenicillin N synthase from Cephalosporium acremonium.

Recombinant isopenicillin N synthase from Cephalosporium acremonium was expressed in Escherichia coli and the protein was purified. After nearly 5000 crystallization trials, the apo enzyme was crystallized by the hanging drop vapour diffusion technique, using polyethylene glycol and lithium sulphate as precipitants. Two crystal forms have been obtained with either octahedral or elongated prismatic habits. The larger octahedral crystals (0.1 mm over-all dimensions) belong to space group I4 with unit cell dimensions of a = b = 124.7 A, c = 156.9 A, and diffract X-rays to about 3.5 A resolution at synchrotrons. The crystallographic asymmetric unit contains a dimer.

Acremonium↗
PubMed1994

A new synthetic ether aminophosphoglyceride exhibits partial modulator activity towards the glucocorticoid receptor.

Modulator is an endogenous low-molecular weight regulator of both glucocorticoid and mineralocorticoid receptors, as well as protein kinase C. Analogs of the putative modulator structure have been synthesized. These compounds include 1-O-(3'-carboxypropyl) or (5'-carboxypentyl)-L-glycero-3-phospho-L-serine or L-threonine, and the D-glycerol stereoisomers. These compounds were tested for in vitro modulator activity using the glucocorticoid-receptor complex activation inhibition and steroid-binding stabilization assays. One of the ether phosphoglycerides, 1-O-(5'-carboxypentyl)-L-glycero-3-phospho-L-threonine (H-GPT-1), partially inhibited steroid-receptor complex activation in a dose-dependent manner. However, none of the other compounds exhibited any modulator activity towards the glucocorticoid-receptor complex. Like modulator, H-GPT-1 did not inhibit activated glucocorticoid-receptor complex binding to DNA-cellulose. Surprisingly, in contrast to modulator, H-GPT-1 partially inhibited unoccupied receptor steroid-binding in a dose-dependent manner. These results suggest that although modulator is not exactly mimicked by this compound, H-GPT-1 is the first synthetic organic molecule to exhibit some modulator activity towards the glucocorticoid receptor.

Adrenalectomy↗
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↗
PubMed1994

On the photochemical release of phosphate from 3,5-dinitrophenyl phosphate in a protein crystal.

In time-resolved diffraction studies, reaction initiation should ideally be both uniform throughout the body of the crystal and rapid with respect to the reaction under study. Caged compounds have been used in a number of experiments to provide photochemical initiation of catalytic reactions in enzyme crystals. No in situ measurements have been reported so far on the kinetics of photolysis or on the distribution of photolysis products within crystals. With the aid of a fast single-crystal microspectrophotometer, we performed quantitative studies on the photolysis of a caged compound, 3,5-dinitrophenyl phosphate, in crystals of glycogen phosphorylase b. The results show that for concentrations required in kinetic experiments, the photolytic release of phosphate from 3,5-dinitrophenyl phosphate is restricted to a thin surface layer only. The liberated substrate is then transported by diffusion into the body of the crystal. In effect, the speed of reaction initiation is limited by the rate of diffusion rather than by the rate of the photochemical reaction. The paper discusses general criteria and experimental strategies for the successful use of photoreactive protective groups in time-resolved diffraction experiments.

Crystallization↗
PubMed1993

Crystallization and preliminary X-ray analysis of the di-haem cytochrome c peroxidase from Pseudomonas aeruginosa.

Cytochrome c551 peroxidase is a periplasmic enzyme expressed in Pseudomonas aeruginosa at low oxygen tensions. The glycosylated enzyme has been purified to homogeneity and crystallized by vapour diffusion techniques using polyethylene glycol 2000 as the precipitant in the presence of isopropanol. The crystals belong to the trigonal space group P3(1)21 or P3(2)21 with unit cell dimensions of a = b = 113.8 A, c = 72.0 A. They are suitable for X-ray analysis and diffract to dmin = 2.5 A. There is one peroxidase molecule in the crystallographic asymmetric unit.

Cytochrome-c Peroxidase↗
PubMed1993

Crystallization and preliminary crystallographic study of cytochrome cd1 nitrite reductase from Thiosphaera pantotropha.

Single crystals of cytochrome cd1 nitrite reductase from Thiosphaera pantotropha have been grown in the presence of ammonium sulphate by vapour diffusion techniques. Crystals are suitable for X-ray analysis and diffract to dmin = 1.5 A. They belong to space group P2(1) with unit cell dimensions of a = 107.8 A, b = 61.9 A, c = 101.2 A, beta = 112.5 degrees. The crystallographic asymmetric unit contains a dimer.

Bacteria, Aerobic↗
PubMed1993

Structure of glycosomal glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma brucei determined from Laue data.

The three-dimensional structure of glycosomal glyceraldehyde-3-phosphate dehydrogenase [D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.12.1.12] from the sleeping-sickness parasite Trypanosoma brucei was solved by molecular replacement at 3.2-A resolution with an x-ray data set collected by the Laue method. For data collection, three crystals were exposed to the polychromatic synchrotron x-ray beam for a total of 20.5 sec. The structure was solved by using the Bacillus stearothermophilus enzyme model [Skarzyński, T., Moody, P. C. E. & Wonacott, A. J. (1987) J. Mol. Biol. 193, 171-187] with a partial data set which was 37% complete. The crystals contain six subunits per asymmetric unit, which allowed us to overcome the absence of > 60% of the reflections by 6-fold density averaging. After molecular dynamics refinement, the current molecular model has an R factor of 17.6%. Comparing the structure of the trypanosome enzyme with that of the homologous human muscle enzyme, which was determined at 2.4-A resolution, reveals important structural differences in the NAD binding region. These are of great interest for the design of specific inhibitors of the parasite enzyme.

Animals↗
PubMed1993

Structure-function relationships of alkyl-lysophospholipid analogs in selective antitumor activity.

This investigation was initiated in order to delineate the structure-function relationship of the anticancer alkyl-lysophospholipids and assess their degree of selective cytotoxicity toward neoplastic cells. A series of glycerol phosphocholine analogs with varying substitutions in the sn-1 and sn-2 position were tested for their inhibitory activity as measured by thymidine incorporation, clonogenic assays and effects on protein kinase C activity against a series of human leukemic cell lines and healthy bone marrow progenitor cells. The IC50 was determined for each of the compounds in each cell line and healthy bone marrow cells following a 4-h incubation. The data indicated that a 16-18 carbon chain at the sn-1 coupled with a short substitution at sn-2 had the broadest antitumor activity and was the least toxic to normal bone marrow cells. The results provide a number of useful leads toward the design and development of potentially more active phospholipid compounds.

Antineoplastic Agents↗