Lars Prade

Implications for the catalytic mechanism of the vanadium-containing enzyme chloroperoxidase from the fungus Curvularia inaequalis by X-ray structures of the native and peroxide form.

Implications for the catalytic mechanism of the vanadium-containing chloroperoxidase from the fungus Curvularia inaequalis have been obtained from the crystal structures of the native and peroxide forms of the enzyme. The X-ray structures have been solved by difference Fourier techniques using the atomic model of the azide chloroperoxidase complex. The 2.03 A crystal structure (R = 19.7%) of the native enzyme reveals the geometry of the intact catalytic vanadium center. The vanadium is coordinated by four non-protein oxygen atoms and one nitrogen (NE2) atom from histidine 496 in a trigonal bipyramidal fashion. Three oxygens are in the equatorial plane and the fourth oxygen and the nitrogen are at the apexes of the bipyramid. In the 2.24 A crystal structure (R = 17.7%) of the peroxide derivate the peroxide is bound to the vanadium in an eta2-fashion after the release of the apical oxygen ligand. The vanadium is coordinated also by 4 non-protein oxygen atoms and one nitrogen (NE2) from histidine 496. The coordination geometry around the vanadium is that of a distorted tetragonal pyramid with the two peroxide oxygens, one oxygen and the nitrogen in the basal plane and one oxygen in the apical position. A mechanism for the catalytic cycle has been proposed based on these X-ray structures and kinetic data.

Staurosporine-induced conformational changes of cAMP-dependent protein kinase catalytic subunit explain inhibitory potential.

BACKGROUND Staurosporine inhibits most protein kinases at low nanomolar concentrations. As most tyrosine kinases, along with many serine/threonine kinases, are either proto oncoproteins or are involved in oncogenic signaling, the development of protein kinase inhibitors is a primary goal of cancer research. Staurosporine and many of its derivatives have significant biological effects, and are being tested as anticancer drugs. To understand in atomic detail the mode of inhibition and the parameters of high-affinity binding of staurosporine to protein kinases, the molecule was cocrystallized with the catalytic subunit of cAMP-dependent protein kinase. RESULTS The crystal structure of the protein kinase catalytic subunit with staurosporine bound to the adenosine pocket shows considerable induced-fit rearrangement of the enzyme and a unique open conformation. The inhibitor mimics several aspects of adenosine binding, including both polar and nonpolar interactions with enzyme residues, and induces conformational changes of neighboring enzyme residues. CONCLUSIONS The results explain the high inhibitory potency of staurosporine, and also illustrate the flexibility of the protein kinase active site. The structure, therefore, is not only useful for the design of improved anticancer therapeutics and signaling drugs, but also provides a deeper understanding of the conformational flexibility of the protein kinase.

Crystal structure of Escherichia coli cystathionine γ-synthase at 1.5 Å resolution

The transsulfuration enzyme cystathionine γ‐synthase (CGS) catalyses the pyridoxal 5′‐phosphate (PLP)‐dependent γ‐replacement of O‐succinyl‐L‐homoserine and L‐cysteine, yielding L‐cystathionine. The crystal structure of the Escherichia coli enzyme has been solved by molecular replacement with the known structure of cystathionine β‐lyase (CBL), and refined at 1.5 Å resolution to a crystallographic R‐factor of 20.0%. The enzyme crystallizes as an α4 tetramer with the subunits related by non‐crystallographic 222 symmetry. The spatial fold of the subunits, with three functionally distinct domains and their quarternary arrangement, is similar to that of CBL. Previously proposed reaction mechanisms for CGS can be checked against the structural model, allowing interpretation of the catalytic and substrate‐binding functions of individual active site residues. Enzyme‐substrate models pinpoint specific residues responsible for the substrate specificity, in agreement with structural comparisons with CBL. Both steric and electrostatic designs of the active site seem to achieve proper substrate selection and productive orientation. Amino acid sequence and structural alignments of CGS and CBL suggest that differences in the substrate‐binding characteristics are responsible for the different reaction chemistries. Because CGS catalyses the only known PLP‐dependent replacement reaction at Cγ of certain amino acids, the results will help in our understanding of the chemical versatility of PLP.

Structures of herbicides in complex with their detoxifying enzyme glutathione S-transferase - explanations for the selectivity of the enzyme in plants.

BACKGROUND Glutathione S-transferases (GSTs) are detoxifying enzymes present in all aerobic organisms. These enzymes catalyse the conjugation of glutathione with a variety of electrophilic compounds. In plants, GSTs catalyse the first step in the degradation of several herbicides, such as triazines and acetamides, thus playing an important role in herbicide tolerance. RESULTS We have solved the structures of GST-I from maize in complex with an atrazine-glutathione conjugate (at 2.8 A resolution) and GST from Arabidopsis thaliana (araGST) in complex with an FOE-4053-glutathione conjugate (at 2.6 A resolution). These ligands are products of the detoxifying reaction and are well defined in the electron density. The herbicide-binding site (H site) is different in the two structures. The architecture of the glutathione-binding site (G site) of araGST is different to that of the previously described structure of GST in complex with two S-hexylglutathione molecules, but is homologous to that of GST-I. CONCLUSIONS Three features are responsible for the differences in the H site of the two GSTs described here: the exchange of hydrophobic residues of different degrees of bulkiness; a slight difference in the location of the H site; and a difference in the degree of flexibility of the upper side of the H site, which is built up by the loop between helices alpha4 and alpha5. Taking these two structures as a model, the different substrate specificities of other plant GSTs may be explained. The structures reported here provide a basis for the design of new, more selective herbicides.

Structures of class pi glutathione S-transferase from human placenta in complex with substrate, transition-state analogue and inhibitor.

BACKGROUND Glutathione S-transferases (GSTs) are detoxification enzymes, found in all aerobic organisms, which catalyse the conjugation of glutathione with a wide range of hydrophobic electrophilic substrates, thereby protecting the cell from serious damage caused by electrophilic compounds. GSTs are classified into five distinct classes (alpha, mu, pi, sigma and theta) by their substrate specificity and primary structure. Human GSTs are of interest because tumour cells show increased levels of expression of single classes of GSTs, which leads to drug resistance. Structural differences between classes of GST can therefore be utilised to develop new anti-cancer drugs. Many mutational and structural studies have been carried out on the mu and alpha classes of GST to elucidate the reaction mechanism, whereas knowledge about the pi class is still limited. RESULTS We have solved the structures of the pi class GST hP1-1 in complex with its substrate, glutathione, a transition-state complex, the Meisenheimer complex, and an inhibitor, S-(rho-bromobenzyl)-glutathione, and refined them to resolutions of 1.8 A, 2.0 A and 1.9 A, respectively. All ligand molecules are well-defined in the electron density. In all three structures, an additionally bound N-morpholino-ethansulfonic acid molecule from the buffer solution was found. CONCLUSIONS In the structure of the GST-glutathione complex, two conserved water molecules are observed, one of which hydrogen bonds directly to the sulphur atom of glutathione and the other forms hydrogen bonds with residues around the glutathione-binding site. These water molecules are absent from the structure of the Meisenheimer complex bound to GST, implicating that deprotonation of the cysteine occurs during formation of the ternary complex which involves expulsion of the inner bound water molecule. The comparison of our structures with known mu class GST structures show differences in the location of the electrophile-binding site (H-site), explaining the different substrate specificities of the two classes. Fluorescence measurements are in agreement with the position of the N-morpholino-ethansulfonic acid, close to Trp28, identifying a possible ligandin-substrate binding site.

Cloning, purification, crystallization, and preliminary X-ray diffraction analysis of cystathionine γ-synthase from E. coli

The Escherichia coli metB gene has been PCR‐extracted from genomic DNA and placed under the control of a tac and a T7 promoter in plasmids pCYB1 and pET22b(+), respectively, to produce overexpressing bacterial strains for the gene product, cystathionine γ‐synthase. Efficient purification procedures have been developed for a C‐terminally intein‐tagged version and the wild‐type target protein, yielding the product in a quantity and homogeneity amenable to high‐resolution single‐crystal X‐ray analysis. Crystals have been obtained in space group P1 with unit cell constants a=82.2 Å, b=84.2 Å, c=116.2 Å, α=107.0°, β=96.3°, γ=108.0°, suggesting eight monomers per asymmetric unit (VM=2.23 Å3/Da). Crystals diffract to beyond 2.6 Å resolution and a data set complete to 2.8 Å resolution has been collected using a rotating anode X‐ray source. A cryogenic buffer system has been developed to allow synchrotron data collection. Patterson self rotation searches reveal the presence of two independent tetramers with local 222 symmetry in an asymmetric unit. The crystallographic results corroborate and extend previous solution studies regarding the quaternary organization of the enzyme.

Herbicide detoxification by glutathione S-transferases as implicated from X-ray structures

Herbicide selectivity is a major factor in agricultural weed control and results from the differential detoxification ability of plant species. The special agronomic value of plant GSTs originates mainly from their metabolic herbicide detoxification properties that enhance the herbicide tolerance of crops. Glutathione S-transferases (GSTs) are a ubiquitous family of multifunctional enzymes involved in the metabolization of a broad variety of xenobiotics (eg herbicides in plants) and reactive endogenous compounds through covalent linkage to glutathione. The metabolization of FOE 5043 results in a GSH conjugate that is subsequently degraded by release of the thiadiazole moiety. The comparison of crystal structures of different GST classes, including plant GSTs, provides a model system to understand active-site interactions on a molecular level. Additional protein structures of three plant GSTs (Arabidopsis thaliana GST, GST I and III from Zea mays var. mutin) in complex with several ligands (S-hexylglutathione, S-lactoylglutathione, and FOE 5043) may be tools to supply detailed knowledge for the rational design of new herbicides and GSTs for selectivity optimization in crops.