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The catalytic activity of the allosteric enzyme pyruvate decarboxylase from yeast is strictly controlled by its own substrate pyruvate via covalent binding at a separate regulatory site. Kinetic studies, chemical modifications, cross-linking, small-angle X-ray scattering, and crystal structure analyses have led to a detailed understanding of the substrate activation mechanism at an atomic level with C221 as the core moiety of the regulatory site. To characterize the individual role of the residues adjacent to C221, we generated variants H92F, H225F, H310F, A287G, S311A, and C221A/C222A. The integrity of the protein structure of the variants was established by small-angle X-ray scattering measurements. The analyses of both steady state and transient kinetic data allowed the identification of the individual roles of the exchanged side chains during allosteric enzyme activation. In each case, the kinetic pattern of activation was modulated but not completely abolished. Despite the crucial role of C221, the covalent binding of pyruvate is not obligate for enzyme activation but is a requirement for a kinetically efficient transition from the inactive to the active state. Moreover, only one of the three histidines guiding the activator molecule to the binding pocket, H310, specifically interacts with C221. H310 stabilizes the thiolate form of C221, ensuring a rapid nucleophilic attack of the thiolate sulfur on C2 of the regulatory pyruvate, thus forming a regulatory dyad. The influence of the other two histidines is less pronounced. Substrate activation is slightly weakened for A287G and significantly retarded for S311A.

We report on a lab-on-a-chip approach for on-site detection of Phytophthora species that allows visual signal readout. The results demonstrate the significance of single-stranded DNA (ssDNA) generation in terms of improving the intensity of the hybridization signal and to improve the reliability of the method. Conventional PCR with subsequent heat denaturation, sodium hydroxide-based denaturation, lambda exonuclease digestion and two asymmetric PCR methods were investigated for the species P. fragariae, P. kernoviae, and P. ramorum. The positioning of the capture probe within the amplified yeast GTP-binding protein (YPT1) target DNA was also of interest because it significantly influences the intensity of the signal. Statistical tests were used to validate the impact of the ssDNA generation methods and the capture-target probe position. The single-stranded target DNA generated by Linear-After-The-Exponential PCR (LATE-PCR) was found to produce signal intensities comparable to post-PCR exonuclease treatment. The LATE-PCR is the best method for the on-site detection of Phytophthora because the enzymatic digestion after PCR is more laborious and time-consuming.

Pyruvate decarboxylase is a key enzyme in organisms whose energy metabolism is based on alcoholic fermentation. The enzyme catalyses the nonoxidative decarboxylation of 2-oxo acids in the presence of the cofactors thiamine diphosphate and magnesium ions. Pyruvate decarboxylase species from yeasts and plant seeds studied to date are allosterically activated by their substrate pyruvate. However, detailed kinetic studies on the enzyme from Neurospora crassa demonstrate for the first time the lack of substrate activation for a yeast pyruvate decarboxylase species. The quaternary structure of this enzyme species is also peculiar because it forms filamentous structures. The complex enzyme structure was analysed using a number of methods, including small-angle X-ray solution scattering, transmission electron microscopy, analytical ultracentrifugation and size-exclusion chromatography. These measurements were complemented by detailed kinetic studies in dependence on the pH. Structured digital abstract • PDC and PDC bind by light scattering (View Interaction: 1, 2) • PDC and PDC bind by cosedimentation in solution (View Interaction: 1, 2) • PDC and PDC bind by electron microscopy (View interaction)

Cysteine synthesis in bacteria and plants is catalyzed by serine acetyltransferase (SAT) and O-acetylserine (thiol)-lyase (OAS-TL), which form the hetero-oligomeric cysteine synthase complex (CSC). In plants, but not in bacteria, the CSC is assumed to control cellular sulfur homeostasis by reversible association of the subunits. Application of size exclusion chromatography, analytical ultracentrifugation, and isothermal titration calorimetry revealed a hexameric structure of mitochondrial SAT from Arabidopsis thaliana (AtSATm) and a 2:1 ratio of the OAS-TL dimer to the SAT hexamer in the CSC. Comparable results were obtained for the composition of the cytosolic SAT from A. thaliana (AtSATc) and the cytosolic SAT from Glycine max (Glyma16g03080, GmSATc) and their corresponding CSCs. The hexameric SAT structure is also supported by the calculated binding energies between SAT trimers. The interaction sites of dimers of AtSATm trimers are identified using peptide arrays. A negative Gibbs free energy (ΔG = -33 kcal mol(-1)) explains the spontaneous formation of the AtCSCs, whereas the measured SAT:OAS-TL affinity (K(D) = 30 nm) is 10 times weaker than that of bacterial CSCs. Free SAT from bacteria is100-fold more sensitive to feedback inhibition by cysteine than AtSATm/c. The sensitivity of plant SATs to cysteine is further decreased by CSC formation, whereas the feedback inhibition of bacterial SAT by cysteine is not affected by CSC formation. The data demonstrate highly similar quaternary structures of the CSCs from bacteria and plants but emphasize differences with respect to the affinity of CSC formation (K(D)) and the regulation of cysteine sensitivity of SAT within the CSC.

The gene yfdU from Escherichia coli encodes a putative oxalyl coenzyme A decarboxylase, a thiamine diphosphate-dependent enzyme that is potentially involved in the degradation of oxalate. The enzyme has been purified to homogeneity. The kinetic constants for conversion of the substrate oxalyl coenzyme A by the enzyme in the absence and presence of the inhibitor coenzyme A, as well as in the absence and presence of the activator adenosine 5′-diphosphate, were determined using a novel continuous optical assay. The effects of these ligands on the solution and crystal structure of the enzyme were studied using small-angle X-ray scattering and X-ray crystal diffraction. Analyses of the obtained crystal structures of the enzyme in complex with the cofactor thiamine diphosphate, the activator adenosine 5′-diphosphate and the inhibitor acetyl coenzyme A, as well as the corresponding solution scattering patterns, allow comparison of the oligomer structures of the enzyme complexes under various experimental conditions, and provide insights into the architecture of substrate and effector binding sites. Structured digital abstract • MINT-7717846: EcODC (uniprotkb:P0AFI0) and EcODC (uniprotkb:P0AFI0) bind (MI:0407) by X-ray scattering (MI:0826) • MINT-7717834: EcODC (uniprotkb:P0AFI0) and EcODC (uniprotkb:P0AFI0) bind (MI:0407) by X-ray crystallography (MI:0114)

Fungal pathogens such as ascomycete Venturia inaequalis, the pathogenic agent of apple scab, cause severe damage to apple trees (Malus domestica Borkh.). The potential use of enzymes with cell wall degrading capacities such as chitinases, s-1,3-glucanases, and cellulases from mycoparasitic Trichoderma ssp. to control fungal plant pathogens has been investigated. Several studies showed an increase of resistance of apple cultivars against Venturia inaequalis by the insertion of genes encoding chitinase from biocontrol fungus Trichoderma atroviride (T. harzianum). Our study is focused on the effect of constitutive expressed T. atroviride genes encoding exochitinase nag70 and endochitinase ech42 in transgenic apple lines, which lead to increased resistance to fungal pathogens, on non-target organisms as mycorrhizal fungi. Therefore we cultivated plants of the apple cv. ‘Pinova’ and plants of two transgenic ‘Pinova’ lines (T386 and T389) in a sand-vermiculite substrate and inoculated them with the AMF Glomus intraradices and G. mosseae. After four months of growth both transgenic lines showed significantly reduced root colonization rates.

Transketolase from baker's yeast is a thiamin diphosphate-dependent enzyme in sugar metabolism that reconstitutes with various analogues of the coenzyme. The methylated analogues (4′-methylamino-thiamin diphosphate and N1′-methylated thiamin diphosphate) of the native cofactor were used to investigate the function of the aminopyrimidine moiety of the coenzyme in transketolase catalysis. For the wild-type transketolase complex with the 4′-methylamino analogue, no electron density was found for the methyl group in the X-ray structure, whereas in the complex with the N1′-methylated coenzyme the entire aminopyrimidine ring was disordered. This indicates a high flexibility of the respective parts of the enzyme-bound thiamin diphosphate analogues. In the E418A variant of transketolase reconstituted with N1′-methylated thiamin diphosphate, the electron density of the analogue was well defined and showed the typical V-conformation found in the wild-type holoenzyme [Lindqvist Y, Schneider G, Ermler U, Sundstrom M (1992) EMBO J11, 2373–2379]. The near-UV CD spectrum of the variant E418A reconstituted with N1′-methylated thiamin diphosphate was identical to that of the wild-type holoenzyme, while the CD spectrum of the variant combined with the unmodified cofactor did not overlap with that of the native protein. The activation of the analogues was measured by the H/D-exchange at C2. Methylation at the N1′ position of the cofactor activated the enzyme-bound cofactor analogue (as shown by a fast H/D-exchange rate constant). The absorbance changes in the course of substrate turnover of the different complexes investigated (transient kinetics) revealed the stability of the α-carbanion/enamine as the key intermediate in cofactor action to be dependent on the functionality of the 4-aminopyrimidine moiety of thiamin diphosphate.

Summary: Purpose: To investigate a possible relation between antiepileptic drugs (AEDs) and the fatty acid composition of membranes. Methods: Fatty acid (FA) composition of erythrocytes was studied in children with epilepsy receiving AED monotherapy. Children taking valproate (VPA, n = 28), carbamazepine (CBZ, n = 17), or phenobarbitone (PB, n = 14) were compared with healthy controls (n = 25). FAs were measured by capillary-gas chromatography (GC-FID). Results: Significant changes (p < 0.05) in the FA composition of membranes were found. In children treated with VPA, C13:0 was decreased (8.2 ± 2% vs. 10.7 ± 4% in controls) and C14:0 increased (1.4 ± 0.5% vs.1 ± 0.5% in controls). C17:0 again was reduced (9.9 ± 4% vs. 13.2 ± 6% in controls), whereas the long-chained acids were enhanced: C18:2n-6 (6 ± 2.4% vs. 3.9 ± 2.5% in controls), and C20:4n-6 (1.9 ± 1.7% vs. 1.4 ± 0.5% in controls). The nonidentified FAs also increased with VPA therapy: 2.5 ± 0.8% versus 1.7 ± 0.9% in controls. Children treated with CBZ showed only minimal changes of FA composition: C13:0 was decreased compared with controls (8 ± 2% vs. 10.7 ± 4%). No changes were seen in patients taking PB. The mean corpuscular volume (MCV) showed important differences between the study groups: MCV was 84.7 ± 6.0 fl during VPA therapy (p < 0.001) and 85.7 ± 4.1 fl with CBZ (p < 0.001). During PB, the MCV increased to 82.87 ± 3.29 fl compared with controls (78.73 ± 4.92 fl; p < 0.01). Conclusions: VPA therapy is associated with changes of the FA composition of membranes, which is not the case with PB therapy. The implications of this finding remain to be established.

The thiamin diphosphate-dependent enzyme indolepyruvate decarboxylase catalyses the formation of indoleacetaldehyde from indolepyruvate, one step in the indolepyruvate pathway of biosynthesis of the plant hormone indole-3-acetic acid. The crystal structure of this enzyme from Enterobacter cloacae has been determined at 2.65 A resolution and refined to a crystallographic R-factor of 20.5% (Rfree 23.6%). The subunit of indolepyruvate decarboxylase contains three domains of open α/β topology, which are similar in structure to that of pyruvate decarboxylase. The tetramer has pseudo 222 symmetry and can be described as a dimer of dimers. It resembles the tetramer of pyruvate decarboxylase from Zymomonas mobilis, but with a relative difference of 20° in the angle between the two dimers. Active site residues are highly conserved in indolepyruvate/pyruvate decarboxylase, suggesting that the interactions with the cofactor thiamin diphosphate and the catalytic mechanisms are very similar. The substrate binding site in indolepyruvate decarboxylase contains a large hydrophobic pocket which can accommodate the bulky indole moiety of the substrate. In pyruvate decarboxylases this pocket is smaller in size and allows discrimination of larger vs. smaller substrates. In most pyruvate decarboxylases, restriction of cavity size is due to replacement of residues at three positions by large, hydrophobic amino acids such as tyrosine or tryptophan.

Enterobacter cloacae, isolated from the rhizosphere of cucumbers, produces large amounts of indole-3-acetic acid. Indolepyruvate decarboxylase, the key enzyme in the biosynthetic pathway of indole-3-acetic acid, catalyses the formation of indole-3-acetaldehyde and carbon dioxide from indole-3-pyruvic acid. The enzyme requires the cofactors thiamine diphosphate and magnesium ions for catalytic activity. Recombinant indolepyruvate decarboxylase was purified from the host Escherichia coli strain JM109. Specificity of the enzyme for the substrates indole-3-pyruvic acid, pyruvic acid, benzoylformic acid, and seven benzoylformic acid analogues was investigated using a continuous optical assay. Stopped-flow kinetic data showed no indication for substrate activation in the decarboxylation reaction of indole-3-pyruvic acid, pyruvic acid or benzoylformic acid. Size exclusion chromatography and small angle X-ray solution scattering experiments suggested the tetramer as the catalytically active state and a pH-dependent subunit association equilibrium. Analysis of the kinetic constants of the benzoylformic acid analogues according to Hansch et al. [Hansch, C., Leo, A., Unger, S.H., Kim, K.H., Nikaitani, D & Lien, E.J. (1973) J. Med. Chem.16, 1207-1216] and comparison with indole-3-pyruvic acid conversion by pyruvate decarboxylases from Saccharomyces cerevisiae and Zymomonas mobilis provided some insight into the catalytic mechanism of indolepyruvate decarboxylase.

The crystal structure of the complex of the thiamine diphosphate dependent tetrameric enzyme pyruvate decarboxylase (PDC) from brewer's yeast strain with the activator pyruvamide has been determined to 2.4 A resolution. The asymmetric unit of the crystal contains two subunits, and the tetrameric molecule is generated by crystallographic symmetry. Structure analysis revealed conformational nonequivalence of the active sites. One of the two active sites in the asymmetric unit was found in an open conformation, with two active site loop regions (residues 104-113 and 290-304) disordered. In the other subunit, these loop regions are well-ordered and shield the active site from the bulk solution. In the closed enzyme subunit, one molecule of pyruvamide is bound in the active site channel, and is located in the vicinity of the thiazolium ring of the cofactor. A second pyruvamide binding site was found at the interface between the Pyr and the R domains of the subunit in the closed conformation, about 10 A away from residue C221. This second pyruvamide molecule might function in stabilizing the unique orientation of the R domain in this subunit which in turn is important for dimer-dimer interactions in the activated tetramer. No difference electron density in the close vicinity of the side chain of residue C221 was found, indicating that this residue does not form a covalent adduct with an activator molecule. Kinetic experiments showed that substrate activation was not affected by oxidation of cysteine residues and therefore does not seem to be dependent on intact thiol groups in the enzyme. The results suggest that a disorder-order transition of two active-site loop regions is a key event in the activation process triggered by the activator pyruvamide and that covalent modification of C221 is not required for this transition to occur. Based on these findings, a possible mechanism for the activation of PDC by its substrate, pyruvate, is proposed.

In the yeast, Saccharomyces cerevisiae, pyruvate decarboxylase (Pdc) is encoded by the two isogenes PDC1 and PDC5. Deletion of the more strongly expressed PDC1 gene stimulates the promoter activity of both PDC1 and PDC5, a phenomenon called Pdc autoregulation. Hence, pdc1Delta strains have high Pdc specific activity and can grow on glucose medium. In this work we have characterized the mutant alleles pdc1-8 and pdc1-14, which cause strongly diminished Pdc activity and an inability to grow on glucose. Both mutant alleles are expressed as detectable proteins, each of which differs from the wild-type by a single amino acid. The cloned pdc1-8 and pdc1-14 alleles, as well as the in-vitro-generated pdc1-51 (Glu51Ala) allele, repressed expression of PDC5 and diminished Pdc specific activity. Thus, the repressive effect of Pdc1p on PDC5 expression seems to be independent of its catalytic activity. A pdc1-8 mutant was used to isolate spontaneous suppressor mutations, which allowed expression of PDC5. All three mutants characterized had additional mutations within the pdc1-8 allele. Two of these mutations resulted in a premature translational stop conferring phenotypes virtually indistinguishable from those of a pdc1Delta mutation. The third mutation, pdc1-803, led to a deletion of two amino acids adjacent to the pdc1-8 mutation. The alleles pdc1-8 and pdc1-803 were expressed in Escherichia coli and purified to homogeneity. In the crude extract, both proteins had 10% residual activity, which was lost during purification, probably due to dissociation of the cofactor thiamin diphosphate (ThDP). The defect in pdc1-8 (Asp291Asn) and the two amino acids deleted in pdc1-803 (Ser296 and Phe297) are located within a flexible loop in the beta domain. This domain appears to determine the relative orientation of the alpha and gamma domains, which bind ThDP. Alterations in this loop may also affect the conformational change upon substrate binding. The mutation in pdc1-14 (Ser455Phe) is located within the ThDP fold and is likely to affect binding and/or orientation of the cofactor in the protein. We suggest that autoregulation is triggered by a certain conformation of Pdc1p and that the mutations in pdc1-8 and pdc1-14 may lock Pdc1p in vivo in a conformational state which leads to repression of PDC5.

The crystal structure of tetrameric pyruvate decarboxylase from Zymomonas mobilis has been determined at 1.9 A resolution and refined to a crystallographic R-factor of 16.2% and Rfree of 19.7%. The subunit consists of three domains, all of the alpha/beta type. Two of the subunits form a tight dimer with an extensive interface area. The thiamin diphosphate binding site is located at the subunit-subunit interface, and the cofactor, bound in the V conformation, interacts with residues from the N-terminal domain of one subunit and the C-terminal domain of the second subunit. The 2-fold symmetry generates the second thiamin diphosphate binding site in the dimer. Two of the dimers form a tightly packed tetramer with pseudo 222 symmetry. The interface area between the dimers is much larger in pyruvate decarboxylase from Z. mobilis than in the yeast enzyme, and structural differences in these parts result in a completely different packing of the subunits in the two enzymes. In contrast to other pyruvate decarboxylases, the enzyme from Z. mobilis is not subject to allosteric activation by the substrate. The tight packing of the dimers in the tetramer prevents large rearrangements in the quaternary structure as seen in the yeast enzyme and locks the enzyme in an activated conformation. The architecture of the cofactor binding site and the active site is similar in the two enzymes. However, the x-ray analysis reveals subtle but significant structural differences in the active site that might be responsible for variations in the biochemical properties in these enzymes.

The nature of the environment of macromolecules influences and determines the state of their overall structure and the extent of binding of specific (cofactors, substrates) or unspecific ligands. How these interactions between enzyme molecules and ligands influence their quaternary structures and, in this way, the realisation of high catalytic activity will be discussed here for the enzyme pyruvate decarboxylase from various organisms: brewer's yeast, brewer's yeast strain, recombinant wild type and site-specific mutants of Saccharomyces cerevisiae, the recombinant wild type of the bacterium Zymomonas mobilis and germinating seeds of the plant Pisum sativum from a structural point of view including both high resolution models from crystal structure analysis and low resolution models from small angle X-ray solution scattering with synchrotron radiation.

We investigated the importance of the interaction between the Nl'-atom of the cofactor thiamine diphosphate and glutamic acid residue 51 in pyruvate decarboxylase (EC 4.1. 1.1). The yeast wild type gene PDCl and the respective mutant genes (E51Q and E51A) were expressed in Escherichia coli. The three enzymes were purified to homogeneity. They comigrated as a single band during silver-stained SDS/PAGE with a molecular mass of 60 000 Da. A molecular mass of 61 200 +/- 200 Da was determined by mass spectrometry for the subunit. The native enzyme is a homotetramer as demonstrated by gel filtration experiments. Near- and far-UV CD spectra showed no significant differences for the apoenzyme of the wild type and the mutants. Slight differences in the rate of thiamine diphosphate binding to the apoprotein component were observed between the wild type and the E51Q PDC by CD spectroscopy. Compared to the wild type enzyme, thiamine diphosphate binding at the E51A mutant apoprotein is very slow. Only 0.04% of the catalytic activity of the wild type enzyme was observed for the E51Q mutant; the E51A mutant has no detectable catalytic activity. The S0.5 value for the substrate pyruvate is increased 33-fold for the E51Q mutant. Substrate activation was observed for both the wild type and the E51Q mutant. The interaction between the N1'-atom of the coenzyme and glutamic acid 51 strongly influences the catalytic activity but only moderately the binding of the cofactor to the apoenzyme and the substrate activation rate.

Thiamin diphosphate (ThDP) is an essential cofactor for a number of enzymes, and especially involved in the nonoxidative decarboxylation of α-keto acids by pyruvate decarboxylase (PDC). Recently the crystal structure of PDC bound ThDP has been determined. Based on these X-ray data MD simulations of the isolated coenzyme as well as of ThDP in its enzymatic environment were performed, using the GROMOS87 software package. For the ThDP-apoenzyme modelling all significant amino acid residues with a cut-off radius less than 8.5 A from the cofactor were taken into account. Because the activity of the coenzyme mainly depends on the formation of a specific structure, the conformational behavior of ThDP and enzyme bound ThDP were investigated within the MD simulations in more detail. Therefore, trajectories of significant structural parameters such as the ring torsion angles ΦT and ΦP as well as essential hydrogen bonds were analyzed by our graphics tool. Moreover, Ramachandran-like plots with respect to the torsion angles ΦT and ΦP were used for the illustration of preferred orientations of the two aromatic rings in ThDP. Finally, MD simulations on ThDP analogs with less or none catalytic activity and apoenzyme mutants were included, in order to get hints of conformational effects and significant interactions in relation to cofactor-apoenzyme binding and the catalytic mechanism.

Summary: Purpose: We present two children who developed a deficiency of factor XIII with valproate (VPA) treatment. This coagulation disorder has not been described in association with VPA treatment in children, and only very recently in one adult patient. Results: Both patients showed recurrent epistaxis as major clinical sign of a combination of decreased coagulation parameters (factor XIII deficiency with thrombocytopenia and decreased von Willebrand factor, respectively). A few days after reduction or withdrawal of VPA treatment, clinical symptoms disappeared, and laboratory findings were within normal range. Conclusions: VPA is known to influence the synthetic function of the liver and the number and function of megakaryocytes. Therefore an alteration of the factor XIII level by VPA is conceivable. Our case reports suggest that bleeding symptoms during VPA treatment may be caused or aggravated by a decreased factor XIII activity. A determination of factor XIII activity should be considered before surgical procedures during VPA treatment to minimize the risk of (severe) postsurgical bleeding complications.

This study investigated the impact of constitutively expressed Trichoderma atroviride genes encoding exochitinase nag70 or endochitinase ech42 in transgenic lines of the apple cultivar Pinova on the symbiosis with arbuscular mycorrhizal fungi (AMF). We compared the exo- and endochitinase activities of leaves and roots from non-transgenic Pinova and the transgenic lines T386 and T389. Local and systemic effects were examined using own-rooted trees and trees grafted onto rootstock M9. Scab susceptibility was also assessed in own-rooted and grafted trees. AMF root colonization was assessed microscopically in the roots of apple trees cultivated in pots with artificial substrate and inoculated with the AMF Glomus intraradices and Glomus mosseae. Own-rooted transgenic lines had significantly higher chitinase activities in their leaves and roots compared to non-transgenic Pinova. Both of the own-rooted transgenic lines showed significantly fewer symptoms of scab infection as well as significantly lower root colonization by AMF. Biomass production was significantly reduced in both own-rooted transgenic lines. Rootstock M9 influenced chitinase activities in the leaves of grafted scions. When grafted onto M9, the leaf chitinase activities of non-transgenic Pinova (M9/Pinova) and transgenic lines (M9/T386 and M9/T389) were not as different as when grown on their own roots. M9/T386 and M9/T389 were only temporarily less infected by scab than M9/Pinova. M9/T386 and M9/T389 did not differ significantly from M9/Pinova in their root chitinase activities, AMF root colonization and biomass. peerReviewed

The reconstitution of pyruvate decarboxylase starts with reversible binding of thiamine diphosphate and Mg2(+)-ions to the apoenzyme, followed by a rate-limiting conformational change to the catalytically active holoenzyme. Investigations with diphospho-esters of 4-methyl-5-(2-hydroxyethyl)thiazolium derivatives have shown that the diphosphate residue of thiamine diphosphate is the most important part of the coenzyme responsible for the first reversible binding step. Methylation of the N1'-atom of the pyrimidine ring of thiamine diphosphate or 4'-oxythiamine diphosphate prevents the coenzyme from binding stably to the apoenzyme, so that the methylated coenzyme displays no coenzyme activity. In contrast, thiamine diphosphate analogues with bulky residues on the neighbouring C2'-atom of the pyrimidine ring form active holoenzyme complexes. This result shows the essential role of the N1'-atom of thiamine diphosphate in stable cofactor binding. The cofactor binding rate to the dimeric and tetrameric apoenzymes indicates that the cofactor is located in the contact regions of the subunits in the tetrameric enzyme.

This version is a preprint from: http://precedings.nature.com/documents/1639/version/1 Kutter, S., Weiss, M. S., Wille, G., Golbik, R., Spinka, M., & König, S. (2008) Covalently bound substrate at the regulatory site triggers allosteric enzyme activation. Nature Precedings hdl.handle.net/10101/npre.2008.1639.1 Published version (peer reviewed): 10.1074/jbc.M806228200 The mechanism by which the enzyme pyruvate decarboxylase from yeast is activated allosterically has been elucidated. A total of seven three-dimensional structures of the enzyme, of enzyme variants or of enzyme complexes from two yeast species (three of them reported here for the first time) provide detailed atomic resolution snapshots along the activation coordinate. The prime event is the covalent binding of the substrate pyruvate to the side chain of cysteine 221, thus forming a thiohemiketal. This reaction causes the shift of a neighbouring amino acid, which eventually leads to the rigidification of two otherwise flexible loops, where one of the loops provides two histidine residues necessary to complete the enzymatically competent active site architecture. The structural data are complemented and supported by kinetic investigations and binding studies and provide a consistent picture of the structural changes, which occur upon enzyme activation.

The thiamin diphosphate (ThDP)-dependent enzyme indolepyruvate decarboxylase (IPDC) is involved in the biosynthetic pathway of the phytohormone 3-indoleacetic acid and catalyzes the nonoxidative decarboxylation of 3-indolepyruvate to 3-indoleacetaldehyde and carbon dioxide. The steady-state distribution of covalent ThDP intermediates of IPDC reacting with 3-indolepyruvate and the alternative substrates benzoylformate and pyruvate has been analyzed by (1)H NMR spectroscopy. For the first time, we are able to isolate and directly assign covalent intermediates of ThDP with aromatic substrates. The intermediate analysis of IPDC variants is used to infer the involvement of active site side chains and functional groups of the cofactor in distinct catalytic steps during turnover of the different substrates. As a result, three residues (glutamate 468, aspartate 29, and histidine 115) positioned perpendicular to the thiazolium moiety of ThDP are involved in binding of all substrates and decarboxylation of the respective tetrahedral ThDP-substrate adducts. Most likely, interactions of these side chains with the substrate-derived carboxylate account for an optimal orientation of the substrate and/or intermediate in the course of carbon-carbon ligation and decarboxylation supporting the suggested least-motion, maximum overlap mechanism. The active site residue glutamine 383, which is located at the opposite site of the thiazolium nucleus as the "carboxylate pocket" (formed by the Glu-Asp-His triad), is central to the substrate specificity of IPDC, probably through orbital alignment. The Glu51-cofactor proton shuttle is, conjointly with the Glu-Asp-His triad, involved in multiple proton transfer steps, including ylide generation, substrate binding, and product release. Studies with para-substituted benzoylformate substrates demonstrate that the electronic properties of the substrate affect the stabilization or destabilization of the carbanion intermediate or carbanion-like transition state and in that way alter the rate dependence on decarboxylation. In conclusion, general mechanistic principles of catalysis of ThDP-dependent enzymes are discussed.

In pyruvate oxidase (POX) from Lactobacillus plantarum, valine 265 participates in binding the cofactor FAD and is responsible for the strained conformation of its isoalloxazine moiety that is visible in the crystal structure of POX. The contrasting effects of the conservative amino acid exchange V265A on the enzyme's catalytic properties, cofactor affinity, and protein structure were investigated. The most prominent effect of the exchange was observed in the 2.2 A crystal structure of the mutant POX. While the overall structures of the wild-type and the variant are similar, flavin binding in particular is clearly different. Local disorder at the isoalloxazine binding site prevents modeling of the complete FAD cofactor and two protein loops of the binding site. Only the ADP moiety shows well-defined electron density, indicating an "anchor" function for this part of the molecule. This notion is corroborated by competition experiments where ADP was used to displace FAD from the variant enzyme. Despite the fact that the affinity of FAD binding in the variant is reduced, the catalytic properties are very similar to the wild-type, and the redox potential of the bound flavin is the same for both proteins. The rate of electron transfer toward the flavin during turnover is reduced to one-third compared to the wild-type, but k(cat) remains unchanged. Redox-triggered FTIR difference spectroscopy of free FAD shows the nu(C(10a)=N(1)) band at 1548 cm(-)(1). In POX-V265A, this band is found at 1538 cm(-)(1) and thus shifted less strongly than in wild-type POX where it is found at 1534 cm(-)(1). Taking these observations together, the conservative exchange V265A in POX has a surprisingly small effect on the catalytic properties of the enzyme, whereas the effect on the three-dimensional structure is rather big.

Kinetic and spectroscopic data indicated that addition of the donor substrate hydroxypyruvate to the thiamin diphosphate (ThDP)-dependent enzyme transketolase (TK) led to the accumulation of the α-carbanion/enamine of (α,β-dihydroxyethyl) ThDP, the key reaction intermediate in enzymatic thiamin catalysis. The three-dimensional structure of this intermediate trapped in the active site of yeast TK was determined to 1.9-Å resolution by using cryocrystallography. The electron density suggests a planar α-carbanion/enamine intermediate having the E -configuration. The reaction intermediate is firmly held in place through direct hydrogen bonds to His-103 and His-481 and an indirect hydrogen bond via a water molecule to His-69. The 4-NH 2 group of the amino-pyrimidine ring of ThDP is within 3 Å distance to the α-hydroxy oxygen atom of the dihydroxyethyl moiety but at an angle unfavorable for a strong hydrogen bond. No structural changes occur in TK on formation of the reaction intermediate, suggesting that the active site is poised for catalysis and conformational changes during the enzyme reaction are not very likely. The intermediate is present with high occupancy in both active sites, arguing against previous proposals of half-of-the-sites reactivity in yeast TK.

The quaternary structures of the thiamine diphosphate dependent enzyme pyruvate decarboxylase (EC 4.1.1.1) from the recombinant wild type of Saccharomycescerevisiae and Zymomonas mobilis and from germinating Pisum sativum seeds were examined by X-ray solution scattering. The dependence of the subunit association equilibrium on the pH and the presence of the cofactors thiamine diphosphate and magnesium ions were compared, and the differences between the catalytic properties of the different enzymes are discussed. The influence of amino acid substitutions at the cofactor binding site of the enzyme from Saccharomyces cerevisiae (E51 is substituted by Q or A and G413 by W) on the subunit association was examined. Low-resolution models of the P. sativum, Z.mobilis, and S. cerevisiae enzymes were evaluated ab initio from the scattering data. The enzyme from the bacterium and yeast appear as a dimer of dimers, whereas the plant enzyme is an octamer formed by two tetramers arranged side-by-side. The shape of the S. cerevisiae enzyme agrees well with the atomic structure in the crystal but suggests that the dimers in the latter should be tilted by approximately 10 degrees. The resulting modification of the atomic structure also yields a significantly better fit to the experimental solution scattering data than that calculated form the original crystallographic model.

The active human immunodeficiency virus type 1 (HIV-1) protease has a homodimeric structure, the subunits are connected by an 'interface' beta-sheet formed by the NH2- and COOH-terminal amino acid segments. Short peptides derived from these segments are able to inhibit the protease activity in the range of micromolar IC50 values. We have further improved the inhibitory power of such peptides by computer modelling. The best inhibitor, the palmitoyl-blocked peptide Pam-Thr-Val-Ser-Tyr-Glu-Leu, has an IC50 value of less than 1 microM. Some of the peptides also showed very good inhibition of the HIV-2 protease. The C-terminal segment of the HIV-1 matrix protein, Acetyl-Gln-Val-Ser-Gln-Asn-Tyr, also inhibits HIV-1 protease. Kinetic studies confirmed the 'dissociative' mechanism of inhibition by the peptides. Depending on the peptide structure and ionic strength, both dimerization inhibition and competitive inhibition were observed, as well as synergistic effects between competitive inhibitors and interface peptides.

With the goal of obtaining inexpensive yet potent anti-AIDS drugs, simple inhibitors of HIV-1 protease were synthesised. The C 2 -symmetrical pseudopeptidic substrate analogues can be prepared as inhibitors for HIV-1 protease starting from symmetrical ketones 3a-d by a facile four-step synthesis. After bromination of 3a-d to α,α'-dibromoketones 4a-d, we synthesised the diamino compounds 6a-c by Gabriel synthesis, which were then coupled with Z-valine to yield inhibitors with a central keto group 2a-c. We also synthesised inhibitors including a central hydroxy group 8a-d a-i by azidation, reduction with LiAlH 4 and coupling of the β,β'-diaminohydroxy compounds with appropriate peptides. The first set of compounds showed only weak inhibition whereas the latter reach K i values of up to 3.0 μM.

Structure and activation pattern of pyruvate decarboxylase (PDC) from yeast was studied by synchrotron radiation X-ray solution scattering. The results give a direct proof that the reversible deactivation of PDC at pH 8.0 is accompanied by the dissociation of the tetrameric holoenzyme into dimeric halves. The kinetics of this process was followed. At pH 6.5 the dimeric halves reassociate to a tetramer even in the absence of cofactors. The changes of the scattering pattern upon binding of the substrate-like activator pyruvamide indicate that the structure expands in the course of the enzyme activation.

The pH dependence of the quaternary structure of pyruvate decarboxylase (EC 4.1.1.1) has recently been discovered [(1990) FEBS Lett. 266, 17–20; (1992) Biochemistry (in press)]. In the present study we have investigated the change in quaternary structure by observing the binding of the cofactor, thiamine pyrophosphate, using 31P NMR spectroscopy. The dissociation of the native tetramers into dimers when increasing the pH coincides with a weaker binding of the cofactor and loss of enzyme activity. The results provide further evidence that thiamine pyrophosphate is bound primarily via the β-phosphate moiety. In addition, a phosphoserine has been discovered in two of the four subunits.

The substrate activation behaviour of pyruvate decarboxylase from germinating seeds of Pisum sativum is characterised kinetically via stopped-flow measurements and discussed with respect to other species. The involvement of SH-groups in this process is demonstrated by reference experiments with chemically modified enzyme.