PubMed Health⌕ Search

Biomedical subjects

H Eklund

Publications and source records attributed to H Eklund.

At least 19 recordsLinked to original sources
PubMed1998

Structure of an aromatic-ring-hydroxylating dioxygenase-naphthalene 1,2-dioxygenase.

BACKGROUND: Pseudomonas sp. NCIB 9816-4 utilizes a multicomponent enzyme system to oxidize naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. The enzyme component catalyzing this reaction, naphthalene 1,2-dioxygenase (NDO), belongs to a family of aromatic-ring-hydroxylating dioxygenases that oxidize aromatic hydrocarbons and related compounds to cis-arene diols. These enzymes utilize a mononuclear non-heme iron center to catalyze the addition of dioxygen to their respective substrates. The present study was conducted to provide essential structural information necessary for elucidating the mechanism of action of NDO. RESULTS: The three-dimensional structure of NDO has been determined at 2.25 A resolution. The molecule is an alpha 3 beta 3 hexamer. The alpha subunit has a beta-sheet domain that contains a Rieske [2Fe-2S] center and a catalytic domain that has a novel fold dominated by an antiparallel nine-stranded beta-pleated sheet against which helices pack. The active site contains a non-heme ferrous ion coordinated by His208, His213, Asp362 (bidentate) and a water molecule. Asn201 is positioned further away, 3.75 A, at the missing axial position of an octahedron. In the Rieske [2Fe-2S] center, one iron is coordinated by Cys81 and Cys101 and the other by His83 and His104. CONCLUSIONS: The domain structure and iron coordination of the Rieske domain is very similar to that of the cytochrome bc1 domain. The active-site iron center of one of the alpha subunits is directly connected by hydrogen bonds through a single amino acid, Asp205, to the Rieske [2Fe-2S] center in a neighboring alpha subunit. This is likely to be the main route for electron transfer.

Amino Acid Sequence↗
PubMed1998

The three-dimensional structures of two toxins from snake venom throw light on the anticoagulant and neurotoxic sites of phospholipase A2.

The three-dimensional structures of the class II anticoagulant phospholipase A2 (PLA2) toxin RVV-VD from the venom of Russell's viper, Vipera russelli russelli, and the class I neurotoxic PLA2 Notechis II-5 from the, Australian tiger snake, Notechis scutatus scutatus, were determined to 2.2 A and 3.0 A resolution, respectively. Both enzymes are monomeric and consist of 121 and 119 residues, respectively. A comparison of ten class I/II PLA2 structures showed, among other differences, that the beta-sheet of these enzymes (residues 76-83) is about 90 degrees less twisted in class I than in class II PLA2s. This, along with the insertion of some residues in the region 57-59 in class I enzymes (the elapid loop), could be the main reason for the significant difference in the anticoagulant and (presynaptic) neurotoxic properties between the two classes of PLA2. It seems apparent from sequence and structural comparisons that the toxic site of PLA2 responsible for the strong anticoagulancy of these toxins consists of a negatively charged part, Glu53, together with a positively charged ridge of lysine residues free for intermolecular interactions. These lysines differ between the two classes of PLA2.

Amino Acid Sequence↗
PubMed1997

Reactivity of the tyrosyl radical of Escherichia coli ribonucleotide reductase -- control by the protein.

Ribonucleotide reductase is a key enzyme for DNA synthesis. Its small component, named protein R2, contains a tyrosyl radical essential for activity. Consequently, radical scavengers are potential antiproliferative agents. In this study, we show that the reactivity of the tyrosyl radical towards phenols, hydrazines, hydroxyurea, dithionite and ascorbate can be finely tuned by relatively small modifications of its hydrophobic close environment. For example, in this hydrophobic pocket, Leu77-->Phe mutation resulted in a protein with a much higher susceptibility to radical scavenging by hydrophobic agents. This might suggest that the protein is flexible enough to allow small molecules to penetrate in the radical site. When mutations keeping the hydrophobic character are brought further from the radical (for example Ile74-->Phe) the reactivity of the radical is instead very little affected. When a positive charge was introduced (for example Ile74-->Arg or Lys) the protein was more sensitive to negatively charged electron donors such as dithionite. These results allow us to understand how tyrosyl radical sites have been optimized to provide a good stability for the free radical.

Amino Acid Substitution↗
PubMed1997

Binding of allosteric effectors to ribonucleotide reductase protein R1: reduction of active-site cysteines promotes substrate binding.

BACKGROUND: Ribonucleotide reductase (RNR) is an essential enzyme in DNA synthesis, catalyzing all de novo synthesis of deoxyribonucleotides. The enzyme comprises two dimers, termed R1 and R2, and contains the redox active cysteine residues, Cys462 and Cys225. The reduction of ribonucleotides to deoxyribonucleotides involves the transfer of free radicals. The pathway for the radical has previously been suggested from crystallographic results, and is supported by site-directed mutagenesis studies. Most RNRs are allosterically regulated through two different nucleotide-binding sites: one site controls general activity and the other controls substrate specificity. Our aim has been to crystallographically demonstrate substrate binding and to locate the two effector-binding sites. RESULTS: We report here the first crystal structure of RNR R1 in a reduced form. The structure shows that upon reduction of the redox active cysteines, the sulfur atom of Cys462 becomes deeply buried. The more accessible Cys225 moves to the former position of Cys462 making room for the substrate. In addition, the structures of R1 in complexes with effector, effector analog and effector plus substrate provide information about these binding sites. The substrate GDP binds in a cleft between two domains with its beta-phosphate bound to the N termini of two helices; the ribose forms hydrogen bonds to conserved residues. Binding of dTTP at the allosteric substrate specificity site stabilizes three loops close to the dimer interface and the active site, whereas the general allosteric binding site is positioned far from the active site. CONCLUSIONS: Binding of substrate at the active site of the enzyme is structurally regulated in two ways: binding of the correct substrate is regulated by the binding of allosteric effectors and binding of the actual substrate occurs primarily when the active-site cysteines are reduced. One of the loops stabilized upon binding of dTTP participates in the formation of the substrate-binding site through direct interaction with the nucleotide base. The general allosteric effector site, located far from the active site, appears to regulate subunit interactions within the holoenzyme.

Allosteric Regulation↗
PubMed1997

The three-dimensional structure of flavodoxin reductase from Escherichia coli at 1.7 A resolution.

Flavodoxin reductase from Escherichia coli is an FAD-containing oxidoreductase that transports electrons between flavodoxin or ferredoxin and NADPH. Together with flavodoxin, the enzyme is involved in the reductive activation of three E. coli enzymes: cobalamin-dependent methionine synthase, pyruvate formate lyase and anaerobic ribonucleotide reductase. An additional function for the oxidoreductase appears to be to protect the bacteria against oxygen radicals. The three-dimensional structure of flavodoxin reductase has been solved by multiple isomorphous replacement, and has been refined at 1.7 A to an R-value of 18.4% and Rfree 24.8%. The monomeric molecule contains one beta-sandwich FAD domain and an alpha/beta NADP domain. The overall structure is similar to other reductases of the NADP-ferredoxin reductase family in spite of the low sequence similarities within the family. Flavodoxin reductase lacks the loop which is involved in the binding of the adenosine moiety of FAD in other FAD binding enzymes of the superfamily but is missing in the FMN binding phthalate dioxygenase reductase. Instead of this loop, the adenine interacts with an extra tryptophan at the C terminus. The FAD in flavodoxin reductase has an unusual bent conformation with a hydrogen bond between the adenine and the isoalloxazine. This is probably the cause of the unusual spectrum of the enzyme. There is a pronounced cleft close to the isoalloxazine that appears to be well suited for binding of flavodoxin/ferredoxin. Two extra short strands of the NADP-binding domain probably act as an anchor point for the binding of flavodoxin.

Adenosine↗
PubMed1997

Crystallographic investigation of the dependence of calcium and phosphate ions for notexin.

The crystal structure of the neurotoxic phospholipase A2, notexin, revealed three binding sites for sulphate ions which were suggested to be phosphate binding sites of importance for the activity of the toxin. The present investigation shows that the sulphate ion bound to the major binding site alters the structure of residues 60-75. In the absence of sulphate and phosphate, the structure of this loop has a conformation which partly resembles the non-neurotoxic PLA2s. The affinity of notexin for phosphate is 17 microM, as measured by the increase in fluorescence at 345 nm. Since the concentrations of phosphate and sulphate ions in blood plasma are 3 and 1 mM, respectively, the binding site must be occupied under physiological conditions. This major sulphate/phosphate binding site explains the specific affinity labelling by pyridoxal phosphate. Pyridoxal phosphate binds to this anion binding site which allows the reaction with Lys-88 or Lys-89. The structure of notexin in the presence and absence of Ca2+ shows only small local structural differences.

Binding Sites↗
PubMed1997

Ribonucleotide reductase--structural studies of a radical enzyme.

Ribonucleotide reductase contains a stable organic free radical essential for its activity located on a tyrosine residue in the small subunit of the enzyme called R2. The substrate binding site is, however, found in the catalytic subunit called R1. A long-range protein-mediated radical transfer pathway appears to be responsible for the delivery of the radical from the tyrosine in R2 to the substrate on R1. The active site is located deep inside the protein in a very stable beta/alpha-barrel structure and a hydrogen bonded system leads from the surface to Cys439 at the active site which is in excellent position to remove a hydrogen from the 3' of the ribose of a bound substrate nucleotide.

Escherichia coli↗
PubMed1996

Crystal structure of Arabidopsis thaliana NADPH dependent thioredoxin reductase at 2.5 A resolution.

Thioredoxin exists in all organisms and is responsible for the hydrogen transfer to important enzymes for ribonucleotide reduction and the reduction of methionine sulphoxide and sulphate. Thioredoxins have also been shown to regulate enzyme activity in plants and are also involved in the regulation of transcription factors and several other regulatory activities. Thioredoxin is reduced by the flavoenzyme thioredoxin reductase using NADPH. We have now determined the first structure of a eukaryotic thioredoxin reductase, from the plant Arabidopsis thaliana, at 2.5 A resolution. The dimeric A. thaliana thioredoxin reductase is structurally similar to that of the Escherichia coli enzyme, and most differences occur in the loops. Because the plant and E. coli enzymes have the same architecture, with the same dimeric structure and the same position of the redox active disulphide bond, a similar mechanism that involves very large domain rotations is likely for the two enzymes. The subunit is divided into two domains, one that binds FAD and one that binds NADPH. The relative positions of the domains in A. thaliana thioredoxin reductase differ from those of the E. coli reductase. When the FAD domains are superimposed, the NADPH domain of A. thaliana thioredoxin reductase must be rotated by 8 degrees to superimpose on the corresponding domain of the E. coli enzyme. The domain rotation we now observe is much smaller than necessary for the thioredoxin reduction cycle.

Amino Acid Sequence↗
PubMed1996

The three-dimensional structure of mammalian ribonucleotide reductase protein R2 reveals a more-accessible iron-radical site than Escherichia coli R2.

The three-dimensional structure of mouse ribonucleotide reductase R2 has been determined at 2.3 A resolution using molecular replacement and refined to an R-value of 19.1% (Rfree = 25%) with good stereo-chemistry. The overall tertiary structure architecture of mouse R2 is similar to that from Escherichia coli R2. However, several important structural differences are observed. Unlike the E. coli protein, the mouse dimer is completely devoid of beta-strands. The sequences differ significantly between the mouse and E. coli R2s, but there is high sequence identity among the eukaryotic R2 proteins, and the identities are localized over the whole sequence. Therefore, the three-dimensional structures of other mammalian ribonucleotide reductase R2 proteins are expected to be very similar to that of the mouse enzyme. In mouse R2 a narrow hydrophobic channel leads to the proposed binding site for molecular oxygen near to the iron-radical site in the interior of the protein. In E. coli R2 this channel is blocked by the phenyl ring of a tyrosine residue, which in mouse R2 is a serine. These structural variations may explain the observed differences in sensitivity to radical scavengers. The structure determination is based on diffraction data from crystals grown at pH 4.7. Unexpectedly, the protein is not iron-free, but contains one iron ion bound at one of the dinuclear iron sites. This ferric ion is bound with partial occupancy and is coordinated by three glutamic acids (one bidentate) and one histidine in a bipyramidal coordination that has a free apical coordination position. Soaking of crystals in a solution of ferrous salt at pH 4.7 increased the occupancy on the already occupied site, but without any detectable binding at the second site.

Animals↗
PubMed1996

The ten-stranded beta/alpha barrel in ribonucleotide reductase protein R1.

The large subunit of ribonucleotide reductase (RNR) contains a ten-stranded beta/alpha barrel of a new type consisting of two antiparallel halves. The two halves of the barrel are pseudo 2-fold-related, have similar folds but different additional intervening secondary structure elements and loops. The inner diameter of the RNR barrel, 15 A to 20 A, is significantly larger than for the (beta alpha)3 barrels. The larger barrel forms a stable framework which holds an inserted hairpin loop rigidly and exposes active site residues at its tip. The barrel organization allows three cysteine residues to be positioned close to each other without forming unfavorable disulfide bridges between Cys439 on the tip of the inserted loop and the redox-active cysteine residues on the barrel strands. Redox-active cysteine residues separated by more than 200 residues are held in close proximity to each other on adjacent barrel strands.

Amino Acid Sequence↗
PubMed1996

Crystal structure of reduced protein R2 of ribonucleotide reductase: the structural basis for oxygen activation at a dinuclear iron site.

BACKGROUND: Ribonucleotide reductases (RNRs) catalyze the formation of the deoxyribonucleotides that are essential for DNA synthesis. The R2 subunit of Escherichia coli RNR is a homodimer containing one dinuclear iron centre per monomer. A tyrosyl radical is essential for catalysis, and is formed via a reaction in which the reduced, diferrous form of the iron centre activates dioxygen. To help understand the mechanism of oxygen activation, we examined the structure of the diferrous form of R2. RESULTS: The crystal structures of reduced forms of both wild type R2 and a mutant of R2 (Ser211--> Ala) have been determined at 1.7 A and 2.2 A resolution, respectively. The diferrous iron centre was compared to the previously determined structure of the oxidized, diferric form of R2. In both forms of R2 the iron centre is coordinated by the same carboxylate dominated ligand sphere, but in the reduced form there are clear conformational changes in three of the carboxylate ligands and the bridging mu-oxo group and two water molecules are lost. In the reduced form of R2 the coordination number decreases from six to four for both ferrous ions, explaining their high reactivity towards dioxygen. The structure of the mutant Ser211--> Ala, known to have impaired reduction kinetics, shows a large conformational change in one of the neighbouring helices although the iron coordination is very similar to the wild type protein. CONCLUSIONS: Carboxylate shifts are often important for carboxylate coordinated metal clusters; they allow the metals to achieve different coordination modes in redox reactions. In the case of reduced R2 these carboxylate shifts allow the formation of accessible reaction sites for dioxygen. The Ser211--> Ala mutant displays a conformational change in the helix containing the mutation, explaining its altered reduction kinetics.

Carboxylic Acids↗
PubMed1996

Crystal structure of cod liver class I alcohol dehydrogenase: substrate pocket and structurally variable segments.

The structural framework of cod liver alcohol dehydrogenase is similar to that of horse and human alcohol dehydrogenases. In contrast, the substrate pocket differs significantly, and main differences are located in three loops. Nevertheless, the substrate pocket is hydrophobic like that of the mammalian class I enzymes and has a similar topography in spite of many main-chain and side-chain differences. The structural framework of alcohol dehydrogenase is also present in a number of related enzymes like glucose dehydrogenase and quinone oxidoreductase. These enzymes have completely different substrate specificity, but also for these enzymes, the corresponding loops of the substrate pocket have significantly different structures. The domains of the two subunits in the crystals of the cod enzyme further differ by a rotation of the catalytic domains by about 6 degrees. In one subunit, they close around the coenzyme similarly as in coenzyme complexes of the horse enzyme, but form a more open cleft in the other subunit, similar to the situation in coenzyme-free structures of the horse enzyme. The proton relay system differs from the mammalian class I alcohol dehydrogenases. His 51, which has been implicated in mammalian enzymes to be important for proton transfer from the buried active site to the surface is not present in the cod enzyme. A tyrosine in the corresponding position is turned into the substrate pocket and a water molecule occupies the same position in space as the His side chain, forming a shorter proton relay system.

Alcohol Dehydrogenase↗
PubMed1995

Proteins.

Explore the source record for details and available documents.

Journal Article↗
PubMed1995

Copper amine oxidase: a novel use for a tyrosine.

The first three-dimensional structure of copper amine oxidase demonstrates that one tyrosine residue is converted into 2,4,5-trihydroxyphenylalanine quinone (TPQ). TPQ binds to copper in the inactive form of the enzyme but not in the active form.

Amine Oxidase (Copper-Containing)↗
PubMed1995

Crystallization and crystallographic investigations of the small subunit of mouse ribonucleotide reductase.

The R2 protein component of mouse ribonucleotide reductase has been obtained from overproducing Escherichia coli bacteria. It has been crystallized using NaCl as precipitant. The crystals are orthorhombic, space group C222(1), with cell dimensions a = 76.9 A, b = 108.9 A, c = 92.7 A and diffract to at least 2.5 A. The asymmetric unit of the crystals contains one monomer. Rotation and translation function searches using a model based on the weakly homologous E. coli R2 gave one significant peak. Rotation about a crystallographic 2-fold axis parallel to the a-axis produces an R2 dimer with dimer interactions very similar to those found for E. coli R2.

Animals↗
PubMed1995

Crystal structure of thioredoxin-2 from Anabaena.

BACKGROUND: Thioredoxins are ubiquitous proteins that serve as reducing agents and general protein disulfide reductases. The structures of thioredoxins from a number of species, including man and Escherichia coli, are known. Cyanobacteria, such as Anabaena, contain two thioredoxins that exhibit very different activities with target enzymes and share little sequence similarity. Thioredoxin-2 (Trx-2) from Anabaena resembles chloroplast type-f thioredoxin in its activities and the two proteins may be evolutionarily related. We have undertaken structural studies of Trx-2 in order to gain insights into the structure/function relationships of thioredoxins. RESULTS: Anabaena Trx-2, like E. coli thioredoxin, consists of a five-stranded beta sheet core surrounded by four alpha helices. The active site includes a conserved disulfide ring (in the sequence 31WCGPC35). An aspartate (E. coli) to tyrosine (Trx-2) substitution alters the position of this disulfide ring relative to the central pleated sheet. However, loss of this conserved aspartate does not affect the disulfide geometry. In the Trx-2 crystals, the N-terminal residues make extensive contacts with a symmetry-related molecule with hydrogen bonds to residues 74-76 mimicking thioredoxin-protein interactions. CONCLUSIONS: The overall three-dimensional structure of Trx-2 is similar to E. coli thioredoxin and other related disulfide oxido-reductases. Single amino acid substitutions around the protein interaction area probably account for the unusual enzymatic activities of Trx-2 and its ability to discriminate between substrate and non-substrate peptides.

Amino Acid Sequence↗
PubMed1995

The structure of a reduced mutant T4 glutaredoxin.

The mutant T4 glutaredoxin where the active site residues Val15 and Tyr16 have been substituted by Gly and Pro, respectively, crystallizes in a form where the active site disulfide is accessible to reagents. Treatment of the crystals with dithiotreitol causes very subtle changes in the overall glutaredoxin structure. The main differences are seen around the active site where the sulfurs of Cys14 and Cys17 move apart slightly.

Bacteriophage T4↗