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H Eklund

Publications and source records attributed to H Eklund.

At least 55 records · Page 3Linked to original sources
PubMed1994

Crystallographic investigations of alcohol dehydrogenases.

The structures of horse liver alcohol dehydrogenase class I in its apoenzyme form and in different ternary complexes have been determined at high resolution. The complex with NAD+ and the substrate analogue pentafluorobenzyl alcohol gives a detailed picture of the interactions in an enzyme-substrate complex. The alcohol is bound to the zinc and positioned so that the hydrogen atom can be directly transferred to the C4 atom of the nicotinamide ring. The structure of cod liver alcohol dehydrogenase with hybrid properties (functionally of class I but structurally overall closer to class III) has been determined by molecular replacement methods to 3 A resolution. Yeast alcohol dehydrogenase has been crystallized, and native data have been collected to 3 A resolution.

Alcohol Dehydrogenase↗
PubMed1993

Crystallization and crystallographic investigations of ribonucleotide reductase protein R1 from Escherichia coli.

Crystals of Escherichia coli ribonucleotide reductase protein R1 have been grown in complex with a synthetic peptide corresponding to the carboxyl end of protein R2. Good quality crystals could only be obtained after improvement of the purification protocol and are of the space group R32 with hexagonal cell axes a = b = 226 A and c = 341 A. They contain 3 subunits per asymmetric unit and diffract to 2.5 A resolution in synchrotron radiation. A multiple isomorphous replacement map at 5.5A, improved by solvent flattening, shows that the dimeric molecules are elongated, about 110 A long. The dimer is thin in the middle around the molecular two-fold axis. The subunit is shaped like a bowl, probably with the active site in its center.

Amino Acid Sequence↗
PubMed1993

An elongated form of T4 glutaredoxin with four extra residues.

Two different forms of T4 glutaredoxin (thioredoxin) arising from the same gene on a multicopy plasmid in an Escherichia coli expression system have been isolated and characterized. Up to one-fourth of purified T4 glutaredoxin has an extension of four amino acids in the carboxy terminus, with the sequence aspartate, arginine, isoleucine, lysine. This four-residue extension may be caused by a translational +1 frameshift at the UGA terminator codon.

Amino Acid Sequence↗
PubMed1993

Structure and function of the Escherichia coli ribonucleotide reductase protein R2.

The crystal structure of the ribonucleotide reductase free radical protein R2 from Escherichia coli has been determined by multiple isomorphous replacement and twofold molecular averaging. The structure has been refined at 2.2 A resolution to R = 0.175. The subunit structure of the R2 protein has a novel fold where the basic motif is a bundle of eight long helices. The R2 dimer has two equivalent dinuclear iron centers. Each iron center is well buried in the subunit. The iron atoms have both histidine and carboxyl acid ligands and are bridged by an oxide ion and the carboxylate group of Glu115. One iron atom is octahedrally coordinated with small deviations from ideal values, while the coordination of the other iron ion is more distorted, mainly due to the fact that Asp84 is a bidental ligand to this iron atom. The oxidation of the enzymatically essential tyrosine residue (Tyr122) and the dinuclear iron center by molecular oxygen is suggested to take part in a suitable conserved oxygen-binding pocket between the iron center and the tyrosine zeta-oxygen 5.3 A away from the closest iron ion. The tyrosine proton can be abstracted by the dioxygen and the deprotonated tyrosine residue is then more easily oxidized to a radical species. Tyr122 is buried inside the protein about 10 A from the surface. This has the consequence that the tyrosyl radical cannot participate directly in hydrogen abstraction from the substrate ribose at the active site of the holoenzyme located on the R1 subunit. The radical must then be indirectly involved in the mechanism of the enzyme and an electron transfer reaction between the active site and the tyrosine must take place. Based on the analysis of the available ribonucleotide reductase sequences, the binding surface for the large ribonucleotide reductase protein R1, and a possible route for an electron transport between the buried radical and this surface is described.

Amino Acid Sequence↗
PubMed1993

Crystal structure analysis of a mutant Escherichia coli thioredoxin in which lysine 36 is replaced by glutamic acid.

The structure of a mutant Escherichia coli thioredoxin with a glutamic acid substituted for a conserved lysine at position 36 adjacent to the active site has been solved using molecular replacement and refined at 2.0-A resolution to a crystallographic residual of 19.9%. The mutant was crystallized in an orthorhombic space group with one molecule in the asymmetric unit. The structure of the mutant thioredoxin shows overall good agreement with the wild-type E. coli thioredoxin. The root-mean-square deviations for all C alpha s are 0.45 and 0.79 A between the mutant structure and the two molecules in the asymmetric unit of the wild-type crystals. Structural changes are seen in several residues in the active-site region preceding the disulfide. A reverse turn of residues 29-32 changes the conformation from a type I to a type II turn. This change may be related to the loss of a hydrogen bond from Lys-36 to the main-chain carbonyl of residue 30 due to the mutation. The C alpha atom of Trp-31 has moved 1.9 A and the indole ring no longer makes hydrogen bonds to the carboxyl group of Asp-61 but instead participates in a crystal contact. The structural differences seen in the mutant thioredoxin may be influenced by the crystal packing. The substituted Glu-36 makes extensive crystal contacts. The static fluorescence of this mutant thioredoxin has a different pH dependence than the wild type.

Binding Sites↗
PubMed1993

Reduction of mutant phage T4 glutaredoxins by Escherichia coli thioredoxin reductase.

Fifteen mutant T4 glutaredoxins (previously T4 thioredoxin) have been assayed for activity with Escherichia coli thioredoxin reductase. The mutations include substitutions in the region of the active site, in the 2 cysteines, and in the 2 residues between the cysteines forming the active-site disulfide bridge. Mutant thioredoxins where substitutions have been made in charged residues around the active site show the biggest differences in activity. The positive residues Lys-13 and Lys-21 were found to be important for efficient binding to thioredoxin reductase. Substitution of the aspartic acid at position 80 with a serine produced a glutaredoxin with superior activity. This mutant glutaredoxin has earlier been shown to be more efficient than the wild type in thiol transferase activity (Nikkola, M., Gleason, F. K., Saarinen, M., Joelson, T., Björnberg, O., and Eklund, H. (1991) J. Biol. Chem. 266, 16105-16112). Even the glutaredoxin P66A, where the active-site cis-proline has been substituted, could be efficiently reduced by thioredoxin reductase. Glutaredoxins lacking one or both cysteines were not active.

Bacteriophage T4↗
PubMed1993

Unusual clustering of carboxyl side chains in the core of iron-free ribonucleotide reductase.

The principal driving forces of protein folding are the burial of hydrophobic residues in the interior of proteins and the exposure of charged residues at the surface. Charged residues are only occasionally found in the interior, where they form hydrogen bonds to oppositely charged residues or main-chain atoms. Ribonucleotide reductase, a key enzyme in DNA synthesis, catalyses the de novo production of deoxyribonucleotide precursors. It is composed of two different dimeric proteins R1 and R2 (refs 3-5). R2 subunits contain buried iron-centres with each centre formed by two ferric ions coordinated by four carboxylates and two histidine ligands. Iron-free R2, apoR2, is a precursor of active R2 and folds into a stable protein which is transformed into active R2 by ferrous ions and molecular oxygen. Here we show that the iron-free protein does not undergo any major structural changes compared with the iron-containing R2. The effect of this is a clustering of four carboxyl side chains in the interior of the subunit, in contrast to the normal distribution of charged residues in proteins.

Amino Acid Sequence↗
PubMed1992

Structure of oxidized bacteriophage T4 glutaredoxin (thioredoxin). Refinement of native and mutant proteins.

The structure of wild-type bacteriophage T4 glutaredoxin (earlier called thioredoxin) in its oxidized form has been refined in a monoclinic crystal form at 2.0 A resolution to a crystallographic R-factor of 0.209. A mutant T4 glutaredoxin gives orthorhombic crystals of better quality. The structure of this mutant has been solved by molecular replacement methods and refined at 1.45 A to an R-value of 0.175. In this mutant glutaredoxin, the active site residues Val15 and Tyr16 have been substituted by Gly and Pro, respectively, to mimic that of Escherichia coli thioredoxin. The main-chain conformation of the wild-type protein is similar in the two independently determined molecules in the asymmetric unit of the monoclinic crystals. On the other hand, side-chain conformations differ considerably between the two molecules due to heterologous packing interactions in the crystals. The structure of the mutant protein is very similar to the wild-type protein, except at mutated positions and at parts involved in crystal contacts. The active site disulfide bridge between Cys14 and Cys17 is located at the first turn of helix alpha 1. The torsion angles of these residues are similar to those of Escherichia coli thioredoxin. The torsion angle around the S-S bond is smaller than that normally observed for disulfides: 58 degrees, 67 degrees and 67 degrees for wild-type glutaredoxin molecule A and B and mutant glutaredoxin, respectively. Each sulfur atom of the disulfide cysteines in T4 glutaredoxin forms a hydrogen bond to one main-chain nitrogen atom. The active site is shielded from solvent on one side by the beta-carbon atoms of the cysteine residues plus side-chains of residues 7, 9, 21 and 33. From the opposite side, there is a cleft where the sulfur atom of Cys14 is accessible and can be attacked by a nucleophilic thiolate ion in the initial step of the reduction reaction.

Amino Acid Sequence↗
PubMed1992

Substitution of manganese for iron in ribonucleotide reductase from Escherichia coli. Spectroscopic and crystallographic characterization.

Each polypeptide chain of protein R2, the small subunit of ribonucleotide reductase from Escherichia coli, contains a stable tyrosyl radical and two antiferromagnetically coupled oxo-bridged ferric ions. A refined structure of R2 has been recently obtained. R2 can be converted into apoR2 by chelating out the metal cofactor and scavenging the radical. This study shows that apoR2 has a very strong affinity for four stable Mn2+ ions. The manganese-containing form of R2, named Mn-R2, has been studied by EPR spectroscopy and x-ray crystallography. It contains two binuclear manganese clusters in which the two manganese ions occupy the natural iron-binding sites and are only bridged by carboxylates from glutamates 115 and 238. This in turn explains why the spin-exchange interaction between the two ions is very weak and why Mn-R2 is EPR active. Mn-R2 could provide a model for the native diferrous form of protein R2, and a detailed molecular mechanism for the reduction of the iron center of protein R2 is proposed.

Amino Acid Sequence↗
PubMed1992

The active site structure of methane monooxygenase is closely related to the binuclear iron center of ribonucleotide reductase.

Methane monooxygenase (MMO) catalyses the biological transformation of methane to methanol at a binuclear iron site. Guided by the three-dimensional structure of the R2 protein of E. coli ribonucleotide reductase (RNR), we have aligned the sequences of two different MMOs with the sequences of the iron coordinating four helix bundle in R2. The model suggests that the central four helix bundle of R2 is present also in MMO. The iron coordination is similar in MMO and R2 with two histidine ligands and four carboxyl ligands in both cases. The residues lining the proposed oxygen binding site in MMO are significantly smaller in MMO than in R2 allowing binding of both molecular oxygen and methane at this site. This binding site is lined by residues Cys151, Thr213, Ile217 and Ile(Val)239.

Amino Acid Sequence↗
PubMed1992

The major piscine liver alcohol dehydrogenase has class-mixed properties in relation to mammalian alcohol dehydrogenases of classes I and III.

The major alcohol dehydrogenase of cod liver has been purified, enzymatically characterized, and structurally analyzed in order to establish original functions and relationships among the deviating classes of the enzyme in mammalian tissues. Interestingly, the cod enzyme exhibits mixed properties--many positional identities with a class III protein, but functionally a class I enzyme--blurring the distinction among the classes of alcohol dehydrogenase. The two domain interfaces, affected by movements upon coenzyme binding, both exhibit substitutions in a manner thus far unique to the cod enzyme. In contrast, coenzyme-binding residues are highly conserved. At the active site, inner and outer parts of the substrate pocket show different extents of amino acid replacement. In total, no less than 7-10 residues of 11 in the substrate binding pocket differ from those of all the mammalian classes, explaining the substrate specificities. However, the inner part of the substrate pocket is very similar to that of the class I enzymes, which is compatible with the observed characteristics of the cod enzyme: ethanol is an excellent substrate (Km = 1.2 mM) and 4-methylpyrazole is a strong inhibitor (Ki = 0.1 microM). These values are about as low as those typical for the ethanol-active class I mammalian enzyme and do not at all resemble those for class III, for which ethanol is hardly a substrate and pyrazole is hardly an inhibitor. Further out in the substrate pocket, several residues differ from the mammalian classes, affecting large substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohol Dehydrogenase↗
PubMed1992

The three-dimensional structure of notexin, a presynaptic neurotoxic phospholipase A2 at 2.0 A resolution.

The three-dimensional structure of notexin has been solved by molecular replacement methods. The structure has been refined at 2.0 A resolution to a crystallographic R-value of 16.5% with good stereo-chemistry. The core of the protein is very similar to other phospholipase A2s (PLA2 s) but several parts of the molecule are distinctly different. The most significant differences from PLA2 s from bovine pancreas and rattlesnake occur in the stretches 56-80 and 85-89. Residue 69, which has been shown to be important for phospholipase binding, has a different conformation and different interactions than in other known PLA2s. The C alpha positions for residues 86-88 differ by about 6 A from both the bovine and the rattlesnake enzyme. The crystals contain no Ca2+ ions. Instead, a water molecule occupies the calcium site.

Amino Acid Sequence↗
PubMed1992

A single-residue exchange gives human recombinant beta beta alcohol dehydrogenase gamma gamma isozyme properties.

Amino acid residue 48 in human alcohol dehydrogenase constitutes one of 21 residue differences between the common, adult-type isozyme subunits beta and gamma. It is at the inner part of the substrate pocket and has been ascribed a role in hydrogen-bond formation with both the substrate and coenzyme. In order to allow direct evaluation of its importance, Thr48 of a recombinant non-acetylated beta subunit was mutated to Ser (as in the gamma subunit) or Ala (as in no native form, and not allowing side-chain hydrogen bonds), and the proteins were expressed in Escherichia coli. The two non-acetylated recombinant proteins, the beta 48T form and the mutant beta 48S, gave enzymatically active enzymes with indistinguishable specific activities towards ethanol, whereas the mutant beta 48A showed no enzymatic activity. The most striking differences between dimers with the beta subunit and the beta 48S subunit (both non-acetylated) were observed with cyclohexanol, hydroxysteroids, methanol and ethanol. With cyclohexanol, the Km was lowered from 11 mM to 280 microM, and the kcat/Km ratio, although still less than that for the gamma gamma isozyme, was increased 80-fold. Similarly, beta 48S could use 3 beta-hydroxy-5 beta-androstan-17-one as substrate, like gamma gamma, although again with a catalytic efficiency much less than that for the gamma gamma isozyme. Furthermore, testosterone inhibited beta 48S to 50% at a concentration of 100 microM, whereas the beta beta form was not inhibited. All these results show that residue 48 is responsible for a large part of the differences between the two isozymes beta beta and gamma gamma of human class-I alcohol dehydrogenase. The form with the inactive beta 48A subunit was possible to purify by AMP-Sepharose chromatography, suggesting the presence of a functional NAD-binding site. The enzymatic measurements, demonstrating a transition from one isozyme activity to that characteristic of another, confirmed that a side-chain hydroxyl in residue 48 is required for activity, and interpretation by computer modelling showed marked differences at the active site.

Alcohol Dehydrogenase↗
PubMed1991

A putative glutathione-binding site in T4 glutaredoxin investigated by site-directed mutagenesis.

A glutathione monomer has been docked into the active site cleft of T4 glutaredoxin (previously called T4 thioredoxin) using molecular graphics. The central part of the cleft is formed by the side chain of Tyr-16 on one side and the residues Thr-64, Met-65, and Pro-66 on the other. The entire glutathione molecule fits well into the cleft. A cis-peptide bond between the residues Met-65 and Pro-66 allows glutathione to bind in an anti-parallel fashion to residues 64-66. Hydrogen bonds can be formed between Met-65 and the glutathione cysteine. This binding positions the glutathione sulfur atom ideally for reaction with the glutaredoxin disulfide. In the model, glutathione can form a hydrogen bond to the hydroxyl group of Tyr-16. Charged interactions at opposite ends of the binding cleft are provided by His-12 and Asp-80. The negatively charged alpha-carboxyl group of glutathione may interact with a positive helix dipole of the protein. Fifteen mutant T4 glutaredoxins have been produced and assayed for glutathione binding by determining thioltransferase activity. Mutant proteins with substitutions in the sides of the cleft (Tyr-16, Pro-66) exhibited the most marked decreases in thioltransferase activity. Mutation of His-12 to a serine decreases the catalytic efficiency whereas substitution of Asp-80 by serine increases the catalytic efficiency. A double mutant, D80S;H12S, has much less affinity for glutathione than either single mutant. Substitution of Cys-14 produces an inactive protein, whereas C17S retains some thioltransferase activity.

Amino Acid Sequence↗