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Y Lindqvist

Publications and source records attributed to Y Lindqvist.

At least 55 records · Page 3Linked to original sources
PubMed1997

Three-dimensional structures of glycolate oxidase with bound active-site inhibitors.

A key step in plant photorespiration, the oxidation of glycolate to glyoxylate, is carried out by the peroxisomal flavoprotein glycolate oxidase (EC 1.1.3.15). The three-dimensional structure of this alpha/beta barrel protein has been refined to 2 A resolution (Lindqvist Y. 1989. J Mol Biol 209:151-166). FMN dependent glycolate oxidase is a member of the family of alpha-hydroxy acid oxidases. Here we describe the crystallization and structure determination of two inhibitor complexes of the enzyme, TKP (3-Decyl-2,5-dioxo-4-hydroxy-3-pyrroline) and TACA (4-Carboxy-5-(1-pentyl)hexylsulfanyl-1,2,3-triazole). The structure of the TACA complex has been refined to 2.6 A resolution and the TKP complex, solved with molecular replacement, to 2.2 A resolution. The Rfree for the TACA and TKP complexes are 24.2 and 25.1%, respectively. The overall structures are very similar to the unliganded holoenzyme, but a closer examination of the active site reveals differences in the positioning of the flavin isoalloxazine ring and a displaced flexible loop in the TKP complex. The two inhibitors differ in binding mode and hydrophobic interactions, and these differences are reflected by the very different Ki values for the inhibitors, 16 nM for TACA and 4.8 microM for TKP. Implications of the structures of these enzyme-inhibitor complexes for the model for substrate binding and catalysis proposed from the holo-enzyme structure are discussed.

Alcohol Oxidoreductases↗
PubMed1997

Circular permutations of natural protein sequences: structural evidence.

Over the past few years, evidence has accumulated that shows that circularly permuted proteins resulting from permutations in their coding genes can indeed occur naturally. In most instances, these circularly permuted amino acid sequences have been detected by sequence alignment of homologous proteins. Circular permutations may escape detection, however, when based on sequence comparisons alone, as recently illustrated by transaldolase, a member of the class I aldolase family.

Bacterial Proteins↗
PubMed1996

Crystal structure of the ternary complex of 1,3,8-trihydroxynaphthalene reductase from Magnaporthe grisea with NADPH and an active-site inhibitor.

BACKGROUND: The enzyme 1,3,8-trihydroxynaphthalene reductase (THNR) catalyzes an essential reaction in the biosynthesis of melanin, a black pigment crucial for the pathogenesis of the rice blast fungus, Magnaporthe grisea. The enzyme is the biochemical target of several commercially important fungicides which are used to prevent blast disease in rice plants. We have determined the structure of the ternary complex of THNR with bound NADPH and a fungicide, tricyclazole. RESULTS: Crystallographic analysis showed four identical subunits of THNR to form a tetramer with 222 symmetry. The enzyme subunit consists of a single domain comprising a seven-stranded beta sheet flanked by eight alpha helices; the subunit contains a dinucleotide-binding fold which binds the coenzyme, NADPH. Tricyclazole, an inhibitor of the enzyme, binds at the active site in the vicinity of the NADPH nicotinamide ring. The active site contains a Ser-Tyr-Lys triad which is proposed to participate in catalysis. Coenzyme specificity is partly conferred by the interaction of a single basic residue, Arg39, with the 2' phosphate group of NADPH. CONCLUSIONS: The structural model reveals THNR to belong to the family of short chain dehydrogenases. Despite the diversity of the chemical reactions catalyzed by this family of enzymes, their tertiary structures are very similar. In particular THNR has many amino acid sequence identities, and thus most probably high structural similarities, to enzymes involved in fungal aflatoxin synthesis. The structure of THNR in complex with NADPH and tricyclazole provides new insights into the structural basis of inhibitor binding. This new information may aid in the design of new inhibitors for rice crop protection.

Amino Acid Sequence↗
PubMed1996

Crystal structure of delta9 stearoyl-acyl carrier protein desaturase from castor seed and its relationship to other di-iron proteins.

The three-dimensional structure of recombinant homodimeric delta9 stearoyl-acyl carrier protein desaturase, the archetype of the soluble plant fatty acid desaturases that convert saturated to unsaturated fatty acids, has been determined by protein crystallographic methods to a resolution of 2.4 angstroms. The structure was solved by a combination of single isomorphous replacement, anomalous contribution from the iron atoms to the native diffraction data and 6-fold non-crystallographic symmetry averaging. The 363 amino acid monomer consists of a single domain of 11 alpha-helices. Nine of these form an antiparallel helix bundle. The enzyme subunit contains a di-iron centre, with ligands from four of the alpha-helices in the helix bundle. The iron ions are bound in a highly symmetric environment, with one of the irons forming interactions with the side chains of E196 and H232 and the second iron with the side chains of E105 and H146. Two additional glutamic acid side chains, from E143 and E229, are within coordination distance to both iron ions. A water molecule is found within the second coordination sphere from the iron atoms. The lack of electron density corresponding to a mu-oxo bridge, and the long (4.2 angstroms) distance between the iron ions suggests that this probably represents the diferrous form of the enzyme. A deep channel which probably binds the fatty acid extends from the surface into the interior of the enzyme. Modelling of the substrate, stearic acid, into this channel places the delta9 carbon atom in the vicinity of one of the iron ions.

Amino Acid Sequence↗
PubMed1996

Crystal structure of transaldolase B from Escherichia coli suggests a circular permutation of the alpha/beta barrel within the class I aldolase family.

BACKGROUND: Transaldolase is one of the enzymes in the non-oxidative branch of the pentose phosphate pathway. It transfers a C3 ketol fragment from a ketose donor to an aldose acceptor. Transaldolase, together with transketolase, creates a reversible link between the pentose phosphate pathway and glycolysis. The enzyme is of considerable interest as a catalyst in stereospecific organic synthesis and the aim of this work was to reveal the molecular architecture of transaldolase and provide insights into the structural basis of the enzymatic mechanism. RESULTS: The three-dimensional (3D) structure of recombinant transaldolase B from E. coli was determined at 1.87 A resolution. The enzyme subunit consists of a single eight-stranded alpha/beta-barrel domain. Two subunits form a dimer related by a twofold symmetry axis. The active-site residue Lys132 which forms a Schiff base with the substrate is located at the bottom of the active-site cleft. CONCLUSIONS: The 3D structure of transaldolase is similar to structures of other enzymes in the class I aldolase family. Comparison of these structures suggests that a circular permutation of the protein sequence might have occurred in transaldolase, which nevertheless results in a similar 3D structure. This observation provides evidence for a naturally occurring circular permutation in an alpha/beta-barrel protein. It appears that such genetic permutations occur more frequently during evolution than was previously thought.

Amino Acid Sequence↗
PubMed1996

Crystallization and preliminary X-ray crystallographic analysis of recombinant transaldolase B from Eschericha coli.

Recombinant transaldolase from Escherichia coli, an enzyme of the pentose phosphate pathway has been crystallized by the vapor-diffusion method using polyethylene glycol 6000 as precipitant. The crystals are orthorhombic, space group P2(1)2(1)2(1) with cell dimensions a = 68.9, b = 91.3 and c = 130.5 A and diffract to 2 A resolution on a conventional X-ray source. The asymmetric unit very likely contains two subunits, corresponding to a packing density of 2.9 A(3) Da(-1).

Journal Article↗
PubMed1996

Crystallization and preliminary x-ray diffraction study of 1 ,3,8-trihydroxynaphthalene reductase from Magnaporthe grisea.

1,3,8-Trihydroxynaphthalene reductase was crystallized in the presence of NADPH and the inhibitor tricyclazole. The crystals are trigonal, space group P3(1)21 or its enantiomorph P3(2)21. Two crystal forms with slightly different cell dimensions were obtained. Form A has unit cell dimensions a = b = 142.6 angstrom, c = 70.1 angstrom and form B cell dimensions a = b = 142.6 angstrom, c = 72.9 angstrom. The diffraction pattern of the latter crystal form extends to 2.5 angstrom resolution.

Ascomycota↗
PubMed1996

High-level expression, purification, and crystallization of recombinant spinach glycolate oxidase in Escherichia coli.

Glycolate oxidase is a flavin-dependent enzyme in the photorespiratory pathway in plants. Here we report the heterologous expression of glycolate oxidase in Escherichia coli and an isolation procedure which results in 4 mg pure protein per gram cell paste in only 1.5 days. This corresponds to a more than 50-fold improvement in yield compared to previously reported expression systems. The purified recombinant protein can be crystallized easily, and the crystals are isomorphous to those obtained from the protein isolated from spinach. The availability of large amounts of enzyme will be a great advantage in the 3D structural and biochemical studies of mutants and inhibitor complexes.

Alcohol Oxidoreductases↗
PubMed1996

Protein-biotin interactions.

The three-dimensional structures of several biotin-binding proteins are now known, giving insights into the molecular architecture of the binding sites for biotin. In combination with biochemical and computational approaches, these structural insights provide the basis for our present understanding of biotin-protein interactions which, in some cases, give rise to spectacular binding constants.

Bacterial Proteins↗
PubMed1995

His103 in yeast transketolase is required for substrate recognition and catalysis.

Crystallographic studies of thiamin-diphosphate-dependent transketolase from Saccharomyces cerevisiae suggested the invariant active-site residue H103 as a possible enzymic group binding the C1 hydroxyl group of the donor substrate and stabilizing the reaction intermediate. To test this hypothesis, H103 was replaced by alanine, asparagine and phenylalanine using site-directed mutagenesis. The crystallographic analysis of the mutant transketolases verified that no structural changes occurred as a consequence of the side-chain replacements. The residual catalytic activities of the mutant enzymes were 4.3% for the H103A, 2.4% for the H103N and 0.1% for the H103F mutant transketolase. Further kinetic analysis of the H103A and H103N mutant enzymes showed that the Km values for the coenzyme were increased by about eightfold. The Km values for the acceptor substrate ribose 5-phosphate were similar to the Km value for wild-type transketolase. However, the Km value for the donor substrate, xylulose 5-phosphate is increased more than tenfold in these two mutants. Circular dichroism spectra of the mutant enzymes also indicated a weaker binding of the donor substrate and/or a less stable reaction intermediate. These observations provide further evidence in support of the proposed role for this invariant residue in recognition of the donor substrate by forming a hydrogen bond between the side chain of H103 and the C1 hydroxyl group of the sugar phosphate. The significant decrease in catalytic activity suggests that this residue also facilitates catalysis, possibly by maintaining the optimal orientation of the donor substrate and reaction intermediates.

Binding Sites↗
PubMed1995

Mechanism of an ATP-dependent carboxylase, dethiobiotin synthetase, based on crystallographic studies of complexes with substrates and a reaction intermediate.

The crystal structures of six complexes of homodimeric Escherichia coli dethiobiotin synthetase with a variety of substrates, substrate analogs, and products have been determined to high resolution. These include (1) the binary complex of dethiobiotin synthetase and the N7-carbamate of 7,8-diaminononanoic acid, (2) the binary complex of enzyme and the alternate substrate, 3-(1-aminoethyl)-nonanedioic acid, (3) the binary complex of enzyme with the product ADP, (4) the quaternary complex of enzyme, ADP, the N7-carbamate of 7,8-diaminononanoic acid, and Ca2+, (5) the ternary complex of enzyme, the ATP analog adenylyl (beta, gamma-methylene)diphosphonate, and the N7-carbamate of 7,8-diaminononanoic acid, and (6) the quaternary complex of enzyme, the ATP analog adenylyl (beta, gamma-methylene)diphosphonate, 7,8-diaminononanoic acid, and Mn2+. One molecule of each substrate binds to one monomer of the enzyme. ADP and the ATP analogue bind to the classical mononucleotide binding fold with the phosphate groups close to the phosphate binding loop Gly8--Thr16 between beta-strand beta 1 and the N-terminus of alpha-helix alpha 1. The adenine ring is bound in a pocket between beta-strands beta 6 and beta 7. In the quaternary complex with Mn2+, the metal binding site is found in the vicinity of the beta- and gamma-phosphate groups. Two oxygen atoms from the phosphates and oxygen atoms from the side chains of Asp54, Thr16, and Glu115 are ligands to the Mn2+ ion in the quaternary complex. In the complex with ADP and the N7-carbamate of 7,8-diaminononanoic acid prepared in the presence of Ca2+ ions, a different metal binding site is found. The Ca2+ ion is coordinated to an oxygen atom of the alpha-phosphate group of the nucleotide, the side chain of Asp54, and solvent molecules. The 7,8-diaminononanoic acid substrate molecule interacts with residues from both subunits, making the dimer the minimal functional unit. The diamino group binds between the loops after beta 2 and beta 4, and the terminal carboxyl group at the hydrophobic tail of the substrate interacts with the amino terminus of helix alpha 5 and with the side chain of Tyr187 in helix alpha 6 of the second subunit at the monomer-monomer interface. Strong additional electron density close to the N7 nitrogen atom of the 7,8-diaminononanoic acid substrate in some complexes indicates that, even in the absence of added bicarbonate in the crystallization mixture, the carbamylated intermediate is formed in the crystal.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Diphosphate↗
PubMed1995

Structural studies on corn nitrate reductase: refined structure of the cytochrome b reductase fragment at 2.5 A, its ADP complex and an active-site mutant and modeling of the cytochrome b domain.

The refined crystal structures of the recombinant cytochrome b reductase fragment of corn (Zea mays) nitrate reductase, its ADP complex and the active-site mutant Cys242Ser are reported here. The native structure has been refined at 2.5 A resolution to a crystallographic R-factor of 18.7% with root-mean-square (r.m.s) deviations from standard bond lengths and angles of 0.013 A and 2.0 degrees. The diffraction pattern of the crystals is highly anisotropic and correction of this effect lowered the crystallographic R-factor by 5% during the refinement. The structure of the enzyme co-crystallized with ADP has been solved at 2.7 A resolution and refined to an R-factor of 18.6% with r.m.s. deviations from standard bond lengths and angles of 0.014 A and 2.1 degrees. It revealed the binding site of the ADP moiety of the NADH cofactor, which is the electron donor for nitrate reduction. Based on this structure, a model of NADH at the active site of the enzyme was built and the implications for electron transfer from NADH to the flavin cofactor are discussed. The crystal structure of an active-site mutant enzyme, Cys242Ser, has been solved by difference Fourier synthesis and refined to an R-factor of 19.0% to 3.0 A resolution with standard deviations of bond lengths and angles of 0.017 A and 2.5 degrees. This structure analysis suggests that the observed decrease in catalytic activity of this mutant might be due to misalignment of the nicotinamide ring in its binding site. A model of the heme-containing domain of nitrate reductase has been built based on the X-ray structure of bovine cytochrome b5 and has been docked with the cytochrome b reductase fragment of nitrate reductase. The model of the complex contains six salt-bridges at the domain-domain interface and a hydrophobic core. In this model, His48, an invariant residue in the cytochrome b reductase family, forms an interaction with the propionic acid group of the D-ring of the heme cofactor. This group is in contact with the C-8 methyl group of the flavin ring. Residues that might influence the redox potential of the flavin cofactor are proposed and their possible role in electron transfer is discussed.

Adenosine Diphosphate↗
PubMed1995

Involvement of Tyr24 and Trp108 in substrate binding and substrate specificity of glycolate oxidase.

Tyr24 and Trp108 are located in the active site of spinach glycolate oxidase. To elucidate their function in substrate binding and catalysis, they were replaced by phenylalanine and serine, respectively. The [Y24F]glycolate oxidase mutant enzyme showed a tenfold higher Km value for glycolate. L-lactate and DL-2-hydroxybutyrate also showed higher Km values, however, the substrate specificity was unchanged as compared to the wild-type enzyme (Km increases in the order glycolate < DL-2-hydroxybutyrate < L-lactate < L-mandelate). The turnover number and the rate of reduction, found to be rate limiting in catalysis, were only slightly affected by the deletion of the hydroxyl group. These findings suggest that Tyr24 is mostly involved in substrate binding. The spectral features of the [Y24F]glycolate oxidase suggest that a fraction (50-80%) of the protein bears a flavin N(5) adduct instead of the oxidized cofactor. Crystals obtained from the isolated [Y24F]glycolate oxidase mutant protein allowed the determination of the three-dimensional structure. Although the structure was low resolution (0.3 nm), it is evident that the structure determined is that of the N(5) adduct species. In addition to the lacking hydroxyl group of Tyr24, we also observed movements of the amino acid side chains of Arg164 and Trp108, the latter replacing a water molecule in the substrate-binding pocket. Other features predominantly found in the class of flavoprotein oxidases, such as stabilization of the covalent N(5)-sulfite adduct and of the paraquinoid form of 8-mercapto-FMN, were found to be conserved. [W108S]Glycolate oxidase, in contrast, showed dramatic effects on both the Km of substrates as well as the turnover number. The Km for glycolate was increased some hundred fold and the turnover number was decreased 500-fold. In addition, it was found that the higher homologs of glycolate, L-lactate and DL-2-hydroxybutyrate had turnover numbers similar to those found with the wild-type enzyme, although the Km values also increased dramatically. These results indicate that Trp108 is of major importance in catalysis and that this residue is involved in determining the substrate specificity of glycolate oxidase.

Alcohol Oxidoreductases↗
PubMed1995

A method for processing diffraction data from twinned protein crystals and its application in the structure determination of an FAD/NADH-binding fragment of nitrate reductase.

A general method to deconvolute oscillation data sets from twinned protein crystals to a corresponding single-crystal data set has been developed and applied to diffraction data measured from crystals of a fragment containing the FAD- and NADH-binding domains of nitrate reductase. The procedure allows straightforward processing of diffraction data from twinned crystals. Typically, R(merge) values of reduced data sets from the nitrate reductase crystals after deconvolution are about 0.06 compared to 0.13 and higher before deconvolution. Based on these deconvoluted data sets, the structure of the FAD- and NADH-binding domains of nitrate reductase could be solved successfully. The result indicates that crystal twinning does not necessarily prevent crystallographic structure determination.

Journal Article↗
PubMed1995

Purification and preliminary X-ray crystallographic studies of recombinant L-ribulose-5-phosphate 4-epimerase from Escherichia coli.

The araD gene from Escherichia coli, coding for L-ribulose-5-phosphate 4-epimerase, was overexpressed and the resulting enzyme was purified to homogeneity. Crystals of L-ribulose-5-phosphate 4-epimerase, obtained with 4.0 M sodium formate as precipitant, belong to space group P4212 with unit cell dimensions a = b = 107.8 A and c = 281.4 A and diffract to at least 2.2 A resolution. Density measurements of these crystals are consistent with eight subunits in the asymmetric unit.

Base Sequence↗
PubMed1995

Three-dimensional model of the alpha-subunit of bacterial luciferase.

The predicted secondary structure of both subunits of bacterial luciferase is in accordance with a regular 8-fold alpha/beta-barrel structure. The 3D profile confirmed that luciferase subunits are compatible with the alpha/beta-barrel despite the absence of sequence similarity with any alpha/beta-barrel protein. The three-dimensional structure of 260 residues of the alpha-chain of luciferase was modeled from coordinates of glycolate oxidase and then energy minimized. The model obtained satisfies the criteria for the structure of a globular protein and is in accordance with known experimental data. From the model it is possible to predict active site residues involved in binding and catalysis. These predictions, and thus also the model, can be tested by protein engineering experiments.

Aldehydes↗
PubMed1994

Analysis of an invariant cofactor-protein interaction in thiamin diphosphate-dependent enzymes by site-directed mutagenesis. Glutamic acid 418 in transketolase is essential for catalysis.

A homologous expression system and a purification protocol for pure, highly active recombinant yeast transketolase have been developed. The invariant transketolase residue Glu418, which forms a hydrogen bond to the N-1' nitrogen atom of the pyrimidine ring of the cofactor thiamin diphosphate has been replaced by glutamine and alanine. Crystallographic analyses of the mutants show that these amino acid substitutions do not induce structural changes beyond the site of mutation. In both cases, the cofactor binds in a manner identical to the wild-type enzyme. Significant differences in the CD spectra of the mutant transketolases compared with the spectrum of wild-type enzyme indicate differences in the electron distribution of the aminopyrimidine ring of the cofactor. The E418Q mutant shows 2% and the E418A mutant shows about 0.1% of the catalytic activity of wild-type enzyme. The affinities of the mutant enzymes for thiamin diphosphate are comparable with wild-type transketolase. The hydrogen bond between the coenzyme and the side chain of Glu418 is thus not required for coenzyme binding but essential for catalytic activity. The results demonstrate the functional importance of this interaction and support the molecular model for cofactor deprotonation, the first step in enzymatic thiamin catalysis.

Animals↗