Crystal versus solution structures of thiamine diphosphate-dependent enzymes

The quaternary structures of the thiamine diphosphate-dependent enzymes transketolase (EC 2.2.1.1; from Saccharomyces cerevisiae), pyruvate oxidase (EC 1.2.3.3; from Lactobacillus plantarum), and pyruvate decarboxylase (EC 4.1.1.1; from Zymomonas mobilis and brewers' yeast, the latter in the native and pyruvamide-activated forms) were examined by synchrotron x-ray solution scattering. The experimental scattering data were compared with the curves calculated from the crystallographic models of these multisubunit enzymes. For all enzymes noted above, except the very compact pyruvate decarboxylase from Z. mobilis, there were significant differences between the experimental and calculated profiles. The changes in relative positions of the subunits in solution were determined by rigid body refinement. For pyruvate oxidase and transketolase, which have tight intersubunit contacts in the crystal, relatively small modifications of the quaternary structure (root mean square displacements of 0.23 and 0.27 nm, respectively) sufficed to fit the experimental data. For the enzymes with looser contacts (the native and activated forms of yeast pyruvate decarboxylase), large modifications of the crystallographic models (root mean square displacements of 0.58 and 1.53 nm, respectively) were required. A clear correlation was observed between the magnitude of the distortions induced by the crystal environment and the interfacial area between subunits.     

Functional Flexibility of the Transketolase Molecule

Transketolase is the simplest representative of the thiamine diphosphate-dependent enzymes. It was the first of these enzymes for which X-ray analysis was performed. Based on the data of X-ray studies and using the mutagenesis technique, the nature of functional groups of the enzyme involved in the interaction with substrates and cofactors and in the coenzyme activation was defined. Thus, considerable achievements have been made in studying the structure of transketolase. However, there is relatively little information on the conformational flexibility of the enzyme molecule while it is functioning, i.e., during its interaction with cofactors and substrates and in the course of intermediate product formation. This review summarizes mainly the results obtained in the author's group, as well as those rare data on this subject that could be found in literature.     

Half-of-the-sites reactivity of transketolase from Saccharomyces cerevisiae

Cleavage by yeast transketolase of the donor substrate, d-xylulose 5-phosphate, in the absence of the acceptor substrate was studied using stopped-flow spectrophotometry. One mole of the substrate was shown to be cleaved in the prestationary phase, leading to the formation of one mole of the reaction product per mole enzyme, which has two active centers. This observation indicates that only one out of the two active centers functions (i.e., binds and cleaves the substrate) at a time. Such half-of-the-sites reactivity of transketolase conforms well with our understanding, proposed previously, that the active centers of the enzyme operate in sequence (in phase opposition): the cleavage of a ketose within one center (first phase of the transketolase reaction) is paralleled by its formation in the other center (glycolaldehyde residue is condensed with the acceptor substrate, and the second stage of the transketolase reaction is thereby completed) [M.V. Kovina, G.A. Kochetov, FEBS Lett. 440 (1998) 81-84].     

Modification of thiamine pyrophosphate dependent enzyme activity by oxythiamine in Saccharomyces cerevisiae cells

Oxythiamine is an antivitamin derivative of thiamine that after phosphorylation to oxythiamine pyro phosphate can bind to the active centres of thiamine-dependent enzymes. In the present study, the effect of oxythiamine on the viability of Saccharomyces cerevisiae and the activity of thiamine pyrophosphate dependent enzymes in yeast cells has been investigated. We observed a decrease in pyruvate decarboxylase specific activity on both a control and an oxythiamine medium after the first 6 h of culture. The cytosolic enzymes transketolase and pyruvate decarboxylase decreased their specific activity in the presence of oxythiamine but only during the beginning of the cultivation. However, after 12 h of cultivation, oxythiamine-treated cells showed higher specific activity of cytosolic enzymes. More over, it was established by SDS-PAGE that the high specific activity of pyruvate decarboxylase was followed by an increase in the amount of the enzyme protein. In contrast, the mitochondrial enzymes, pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes, were inhibited by oxythiamine during the entire experiment. Our results suggest that the observed strong decrease in growth rate and viability of yeast on medium with oxythiamine may be due to stronger inhibition of mitochondrial pyruvate dehydrogenase than of cytosolic enzymes.     

Glycolytic metabolism in cultured cells of the nervous system IV. The effects of thiamine deficiency on thiamine levels,metabolites and thiamine-dependent enzymes of the C-6 glioma and C-1300 neuroblastoma cell lines.

Summary C-6 glioma and C-1300 neuroblastoma cells were cultured in thiamine deficient and control media. Thiamine levels, transketolase and pyruvate decarboxylase activities, and high energy phosphate metabolites were all measured in deficient and control cells. Thiamine levels in the deficient cells were found to be below the level of detectability. Pyruvate decarboxylase activity was more susceptible to thiamine deficiency in both cell lines than transketolase. In spite of the large decrease in pyruvate decarboxylase activity, high energy phosphate metabolites were not decreased in either cell line. These data indicate that C-6 glioma and C-1300 neuroblastoma cells have the capacity to maintain normal energy metabolites in the presence of large changes in thiamine levels and thiamine dependent enzyme activity.Supported in part by USPHS grant AA 01391.     

Effect of Transketolase Substrates on Holoenzyme Reconstitution and Stability

The influence of transketolase substrates on the interaction of apotransketolase with its coenzyme thiamine diphosphate (TDP) and on the stability of the reconstituted holoenzyme was studied. Donor substrates increased the affinity of the coenzyme for transketolase, whereas acceptor substrate did not. In the presence of magnesium ions, the active centers of transketolase initially identical in TDP binding lose their equivalence in the presence of donor substrates. The stability of transketolase depended on the cation type used during its reconstitution--the holoenzyme reconstituted in the presence of calcium ions was more stable than the holoenzyme produced in the presence of magnesium ions. In the presence of donor substrate, the holoenzyme stability increased without depending on the cation used during the reconstitution. Donor substrate did not influence the interaction of apotransketolase with the inactive analog of the coenzyme N3'-pyridyl thiamine diphosphate and did not stabilize the transketolase complex with this analog. The findings suggest that the effect of the substrate on the interaction of the coenzyme with apotransketolase and on stability of the reconstituted holoenzyme is caused by generation of 2-(alpha,beta-dihydroxyethyl)thiamine diphosphate (an intermediate product of the transketolase reaction), which has higher affinity for apotransketolase than TDP.     

Optical Characteristics of Thiamine in Model Systems and in Holoenzyme

The optical properties of thiamine diphosphate-dependent enzymes change significantly on their interaction with cofactors (thiamine, bivalent metal ions) and substrates. These changes are connected with structural alterations of the active site and the mechanism of its functioning, and in some cases they reflect changes in the optical properties of the coenzyme itself within the protein. The use of optical characteristics, especially together with model systems, appeared to be a rather promising approach for investigation of the active site of thiamine diphosphate-dependent enzymes and the mechanism of its functioning. So, it seemed to be useful to summarize the literature data concerning the optical characteristics of thiamine (thiamine diphosphate) in model systems and the efficiency of their application for study of thiamine diphosphate-dependent enzymes.     

Comparative aspects of hydroxamic acid production by thiamine-dependent enzymes

The reactions of 4-chloronitrosobenzene with pyruvate decarboxylase and transketolase were investigated by use of a new high-pressure liquid chromatography method to determine any differences between these two enzymes with respect to hydroxamic acid production. In addition to the previously established difference in the type of hydroxamic acid produced by the two enzymes, several new and interesting differences in their reaction with nitrosoaromatics were discovered. Most notable was the finding that pyruvate decarboxylase gave 4-chlorophenylhydroxylamine as the major product from 4-chloronitrosobenzene, while transketolase did not produce any detectable hydroxylamine. A redox mechanism was proposed to account for arylhydroxylamine production by pyruvate decarboxylase. This redox mechanism can also explain hydroxamic acid production by pyruvate decarboxylase; however, a previously proposed nucleophilic reaction mechanism occurring simultaneously could not be totally disproven. Either of the two mechanisms is equally likely for transktolase action in view of the present evidence. Another major difference between these enzymes is that the rate of 4-chloronitrosobenzene conversion was found to be much faster for pyruvate decarboxylase than for transketolase when each enzyme was subjected to its own optimal reaction conditions. Transketolase displayed typical enzyme saturation kinetics with 4-chloronitrosobenzene with a K_m of 0.31 mM and V_max of 0.033 μmol ml^?1 min^?1 unit^?1 relative to 5 mMd-fructose 6-phosphate as sugar substrate. On the other hand, the reaction with pyruvate decarboxylase was first order in 4-chloronitrosobenzene with a combined rate constant of 2.0 min^?1 unit^?1 ml.     

New evidence for cofactor's amino group function in thiamin catalysis by transketolase

Transketolase from Saccharomyces cerevisiae exhibits a rarely reported activity with a methylated analogue of the native cofactor, 4′-methylamino-thiamin diphosphate. We demonstrated the kinetic stability of the dihydroxyethyl carbanion/enamine intermediate to be dependent on the functionality of the 4′-aminopyrimidine moiety of thiamin diphosphate [R. Golbik, L.E. Meshalkina, T. Sandalova, K. Tittmann, E. Fiedler, H. Neef, S. König, R. Kluger, G.A. Kochetov, G. Schneider, G. Hübner, Effect of coenzyme modification on the structural and catalytic properties of wild-type transketolase and of the variant E418A from Saccharomyces cerevisae, FEBS J. (2005) 272 1326-1342]. This paper extends these investigations of the function of the coenzyme’s aminopyrimidine in transketolase catalysis exemplified for the 4′-monomethylamino-thiamin diphosphate analogue. Here, we report near UV circular dichroism data and NMR-based analysis of reaction intermediates that give evidence for a strong destabilisation of the carbanion/enamine of DHE-4’-monomethylamino-thiamin diphosphate on the enzyme. A new negative band in near UV circular dichroism arising during turnover is attributed to the conjugate acid of the carbanion/enamine intermediate, an assignment additionally corroborated by ¹H NMR-based intermediate analysis. As opposed to the kinetically stabilized carbanion/enamine intermediate in transketolase when reconstituted with the native cofactor, DHE-4′-monomethylamino-thiamin diphosphate is rapidly released from the active centers during turnover and accumulates in the medium on a preparative scale.     

Amino acids allosterically regulate the thiamine diphosphate-dependent alpha-keto acid decarboxylase from Mycobacterium tuberculosis

The gene rv0853c from Mycobacterium tuberculosis strain H37Rv codes for a thiamine diphosphate-dependent alpha-keto acid decarboxylase (MtKDC), an enzyme involved in the amino acid degradation via the Ehrlich pathway. Steady state kinetic experiments were performed to determine the substrate specificity of MtKDC. The mycobacterial enzyme was found to convert a broad spectrum of branched-chain and aromatic alpha-keto acids. Stopped-flow kinetics showed that MtKDC is allosterically activated by alpha-keto acids. Even more, we demonstrate that also amino acids are potent activators of this thiamine diphosphate-dependent enzyme. Thus, metabolic flow through the Ehrlich pathway can be directly regulated at the decarboxylation step. The influence of amino acids on MtKDC catalysis was investigated, and implications for other thiamine diphosphate-dependent enzymes are discussed.     

Fermentative glycolysis with purified Escherichia coli enzymes for in vitro ATP production and evaluating an engineered enzyme

Each of the twelve enzymes for glycolytic fermentation, eleven from Escherichia coli and one from Saccharomyces cerevisiae, have been over-expressed in E. coli and purified with His-tags. Simple assays have been developed for each enzyme and they have been assembled for fermentation of glucose to ethanol. Phosphorus-31 NMR revealed that this in vitro reaction accumulates fructose 1,6-bisphosphate while recycling the cofactors NAD⁺ and ATP. This reaction represents a defined ATP-regeneration system that can be tailored to suit in vitro biochemical reactions such as cell-free protein synthesis. The enzyme from S. cerevisiae, pyruvate decarboxylase 1 (Pdc1; EC 4.1.1.1), was identified as one of the major ‘flux controlling’ enzymes for the reaction and was replaced with an evolved version of Pdc1 that has over 20-fold greater activity under glycolysis reaction conditions. This substitution was only beneficial when the ratio of glycolytic enzymes was adjusted to suit greater Pdc1 activity.     

Characterization of a glyphosate-insensitive 5-enolpyruvylshikimic acid-3-phosphate synthase

A glyphosate (N-[phosphonomethyl]glycine)-insensitive 5-enolpyruvylshikimic acid-3-phosphate (EPSP) synthase has been purified from a strain of Klebsiella pneumoniae which is resistant to this herbicide [(1984) Arch. Microbiol. 137, 121-123] and its properties compared with those of the glyphosate-sensitive EPSP synthase of the parent strain. The apparent K_m values of the insensitive enzyme for phosphoenolpyruvate (PEP) and shikimate 3-phosphate (S-3-P) were increased 15.6- and 4.3-fold, respectively, as compared to those of the sensitive enzyme, and significant differences were found for the optimal pH and temperature, as well as the isoelectric points of the two enzymes. While PEP protected both enzymes against inactivation by N-ethylmaleimide, 3-bromopyruvate, and phenylglyoxal, glyphosate protected only the sensitive enzyme.     

Poster Sessions BP01: Energy Metabolism. NMR investigation of metabolic alterations in the brain of thiamine‐deficient rats

Thiamine deficiency (TD) results in region‐selective impairment of brain metabolism. Since thiamine is a cofactor for enzymes involved in glucose metabolism, ¹H and ¹³C‐NMR was used to investigate metabolic fluxes through the major pathways of glucose metabolism in vulnerable (medial thalamus, MT; inferior colliculus, IC) and nonvulnerable brain structures of rats made thiamine deficient following treatment with the central thiamine antagonist pyrithiamine vs. pair‐fed controls. Symptomatic stages of TD resulted in decreased glutamate and GABA in MT an IC confirming previous biochemical studies. ¹³C‐isotopomer analysis revealed decreased de novo synthesis of [4–¹³C]glutamate (30%p < 0.02) and [2–¹³C]GABA (60%p < 0.01) in MT and IC consistent with decreased activities of pyruvate‐ and α‐ketoglutarate dehydrogenases. These changes were accompanied by decreased consumption of glucose and increased synthesis of lactate from [1–¹³C]glucose confirming decreased mitochondrial metabolism. Accumulation of glyceraldehyde‐3‐phosphate suggested inhibition of glucose flux through the thiamine‐deficient enzyme transketolase. Onset of symptoms of TD and significant cell death was accompanied by decreased neuronal marker molecules NAA and NAAG in MT. Focal lactate accumulation resulting from decreased activities of mitochondrial thiamine‐dependent enzymes appears to play a key role in the pathogenesis of selective neuronal cell death in TD. [funded by CIHR Canada].     

Molecular-kinetic parameters of thiamine enzymes and the mechanism of antivitamin action of hydroxythiamine in animal organisms]

The molecula-kinetic parameters (Km, Ki) of three thiamine enzymes, e. g. thiamine pyrophosphokinase (EC 2.7.6.2), pyruvate dehydrogenase (EC 1.2.4.1) and transketolase (EC 2.2.1.1) with respect to the effects of the thiamine antimetabolite hydroxythiamine in the whole animal organism have been compared. It has been shown that only the first two enzymes, which interact competitively with the vitamin, antivitamin or their pyrophosphate ethers, obey the kinetic parameters obtained for the purified enzymes in vitro. The anticoenzymic effect of hydroxythiamine pyrophosphate with respect to transketolase is not observed in vivo at maximal concentration of the anticoenzyme in tissues due to the absence of competitive interactions with thiamine pyrophosphate. The incorporation of the true and false coenzymes into transketolase occurs only during de novo transketolase synthesis (the apoform is absent in tissues, with the exception of erythrocytes) and proceeds slowly with a half-life time equal to 24--30 hrs. After a single injection of hydroxythiamine at a large dose (70--400 mg/kg) the maximal inhibition of the transketolase activity in tissues (liver, heart, kidney, muscle, spleen, lungs adrenal grands) manifests itself by the 48th--72nd hour, when the concentration of free hydroxythiamine and its pyrophosphate is minimal and the whole anticoenzyme is tightly bound to the protein, forming the false holoenzyme. The use of hydroxythiamine for inhibition of pyruvate dehydrogenase or transketolase in animal organism is discussed.     

Effects of substitution of aspartate-440 and tryptophan-487 in the thiamin diphosphate binding region of pyruvate decarboxylase from Zymomonas mobilis.

A tryptophan residue at position 487 in Zymomonas mobilis pyruvate decarboxylase was altered to leucine by site-directed mutagenesis. This modified Z. mobilis pyruvate decarboxylase was active when expressed in Escherichia coli and had unchanged kinetics towards pyruvate. The enzyme showed a decreased affinity for the cofactors with the half-saturating concentrations increasing from 0.64 to 9.0 microM for thiamin diphosphate and from 4.21 to 45 microM for Mg2+. Unlike the wild-type enzyme, there was little quenching of tryptophan fluorescence upon adding cofactors to this modified form. The data suggest that tryptophan-487 is close to the cofactor binding site but is not required absolutely for pyruvate decarboxylase activity. Substitution of asparagine, threonine or glycine for aspartate-440, a residue which is conserved between many thiamin diphosphate-dependent enzymes, completely abolishes enzyme activity.     

Kinetic Studies of Mouse Brain Transketolase

Abstract: The activity of transketolase in mouse brain was 5.7 nmol/min/mg protein measured by an enzyme-coupled spectrophotometric assay. The apparent K_m for ribose-5-phosphate was 330 μ M , for d -xylulose-5-phosphate was 120 μ M , and for thiamine pyrophosphate was 7 μ M . However, thiamine pyrophosphate remained tightly bound to transketolase in homogenates in which it dissociated completely from another thiamine pyrophosphate- dependent enzyme, the pyruvate dehydrogenase complex. These data suggest that loss of transketolase activity is likely to be a later consequence of thiamine deficiency in mammalian brain than is decreased activity of pyruvate dehydrogenase complex.     

The crystal structure of phenylpyruvate decarboxylase from Azospirillum brasilense at 1.5 A resolution. Implications for its catalytic and regulatory mechanism

Phenylpyruvate decarboxylase (PPDC) of Azospirillum brasilense, involved in the biosynthesis of the plant hormone indole-3-acetic acid and the antimicrobial compound phenylacetic acid, is a thiamine diphosphate-dependent enzyme that catalyses the nonoxidative decarboxylation of indole- and phenylpyruvate. Analogous to yeast pyruvate decarboxylases, PPDC is subject to allosteric substrate activation, showing sigmoidal v versus [S] plots. The present paper reports the crystal structure of this enzyme determined at 1.5 A resolution. The subunit architecture of PPDC is characteristic for other members of the pyruvate oxidase family, with each subunit consisting of three domains with an open alpha/beta topology. An active site loop, bearing the catalytic residues His112 and His113, could not be modelled due to flexibility. The biological tetramer is best described as an asymmetric dimer of dimers. A cysteine residue that has been suggested as the site for regulatory substrate binding in yeast pyruvate decarboxylase is not conserved, requiring a different mechanism for allosteric substrate activation in PPDC. Only minor changes occur in the interactions with the cofactors, thiamine diphosphate and Mg2+, compared to pyruvate decarboxylase. A greater diversity is observed in the substrate binding pocket accounting for the difference in substrate specificity. Moreover, a catalytically important glutamate residue conserved in nearly all decarboxylases is replaced by a leucine in PPDC. The consequences of these differences in terms of the catalytic and regulatory mechanism of PPDC are discussed.     

Paraquat and menadione exposure of rainbow trout (Oncorhynchus mykiss)--studies of effects on the pentose-phosphate shunt and thiamine levels in liver and kidney

Possible xenobiotic interactions with thiamine were studied in salmonid fish, by repeatedly injecting two model substances, paraquat and menadione, into juvenile rainbow trout (Oncorhynchus mykiss). These two substances were chosen because of their well-known ability to redox-cycle and cause depletion of NADPH in several biological systems. Depletion of NADPH increases metabolism through the pentose-phosphate shunt and may thereby increase the need for thiamine diphosphate by heightened transketolase activity. A special food was produced with lower thiamine content than commercial food, usually enriched with thiamine, which could mask an effect on the thiamine level. After 9 weeks of exposure, glucose-6-phosphate dehydrogenase, transketolase, glutathione reductase and ethoxyresorufin O-deethylase were analysed in liver and kidney cellular sub-fractions as well as analysis of total thiamine concentrations in liver, kidney and muscle. The results showed that paraquat caused a large increase in hepatic glutathione reductase activity and induced hepatic glucose-6-phosphate dehydrogenase activity, i.e., the rate-limiting enzyme in the oxidative part of the pentose-phosphate shunt. Despite this paraquat exposure did not affect transketolase activity and total thiamine concentration.     

Flexibility of thiamine diphosphate revealed by kinetic crystallographic studies of the reaction of pyruvate-ferredoxin oxidoreductase with pyruvate

Pyruvate-ferredoxin oxidoreductases (PFOR) are unique among thiamine pyrophosphate (ThDP)-containing enzymes in giving rise to a rather stable cofactor-based free-radical species upon the decarboxylation of their first substrate, pyruvate. We have obtained snapshots of unreacted and partially reacted (probably as a tetrahedral intermediate) pyruvate-PFOR complexes at different time intervals. We conclude that pyruvate decarboxylation involves very limited substrate-to-product movements but a significant displacement of the thiazolium moiety of ThDP. In this respect, PFOR seems to differ substantially from other ThDP-containing enzymes, such as transketolase and pyruvate decarboxylase. In addition, exposure of PFOR to oxygen in the presence of pyruvate results in significant inhibition of catalytic activity, both in solution and in the crystals. Examination of the crystal structure of inhibited PFOR suggests that the loss of activity results from oxime formation at the 4' amino substituent of the pyrimidine moiety of ThDP.     

Crystal structure of Bifidobacterium Longum phosphoketolase; key enzyme for glucose metabolism in Bifidobacterium

The crystal structure of Bifidobacterium longum phosphoketolase, a thiamine diphosphate (TPP) dependent enzyme, has been determined at 2.2 Å resolution. The enzyme is a dimer with the active sites located at the interface between the two identical subunits with molecular mass of 92.5 kDa. The bound TPP is almost completely shielded from solvent except for the catalytically important C2-carbon of the thiazolium ring, which can be accessed by a substrate sugar through a narrow funnel-shaped channel. In silico docking studies of B. longum phosphoketolase with its substrate enable us to propose a model for substrate binding.

Structured summary

MINT-7985878: PKT (uniprotkb:Q6R2Q7) and PKT (uniprotkb:Q6R2Q7) bind (MI:0407) by X-ray crystallography (MI:0114)