Molecular mechanism of allosteric substrate activation in a thiamine diphosphate-dependent decarboxylase

Thiamine diphosphate-dependent enzymes are involved in a wide variety of metabolic pathways. The molecular mechanism behind active site communication and substrate activation, observed in some of these enzymes, has since long been an area of debate. Here, we report the crystal structures of a phenylpyruvate decarboxylase in complex with its substrates and a covalent reaction intermediate analogue. These structures reveal the regulatory site and unveil the mechanism of allosteric substrate activation. This signal transduction relies on quaternary structure reorganizations, domain rotations, and a pathway of local conformational changes that are relayed from the regulatory site to the active site. The current findings thus uncover the molecular mechanism by which the binding of a substrate in the regulatory site is linked to the mounting of the catalytic machinery in the active site in this thiamine diphosphate-dependent enzyme.     

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.     

Structure and functioning mechanism of transketolase

Studies of thiamine diphosphate-dependent enzymes appear to have commenced in 1937, with the isolation of the coenzyme of yeast pyruvate decarboxylase, which was demonstrated to be a diphosphoric ester of thiamine. For quite a long time, these studies were largely focused on enzymes decarboxylating α-keto acids, such as pyruvate decarboxylase and pyruvate dehydrogenase complexes. Transketolase, discovered independently by Racker and Horecker in 1953 (and named by Racker) [1], did not receive much attention until 1992, when crystal X-ray structure analysis of the enzyme from Saccharomyces cerevisiae was performed [2]. These data, together with the results of site-directed mutagenesis, made it possible to understand in detail the mechanism of thiamine diphosphate-dependent catalysis. Some progress was also made in studies of the functional properties of transketolase. The last review on transketolase, which was fairly complete, appeared in 1998 [3]. Therefore, the publication of this paper should not seem premature.     

Vitamin B1: Metabolism and functions

The review highlights metabolism and biological functions of vitamin B 1 (thiamine). It considers thiamine transport systems in various organisms enzymes of its biosynthesis and degradation, as well as molecular basis of thiamine-dependent hereditary pathologies. A special attention is paid to discussion of the role of thiamine triphosphate and adenylated thiamine triphosphate, a new thiamine derivative recently discovered in living cells.     

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.     

Reversibility of thiamine deficiency-induced partial necrosis and mitochondrial uncoupling by addition of thiamine to neuroblastoma cell suspensions

Culture of neuroblastoma cells in the presence of low thiamine concentration (6 nM) and of the transport inhibitor amprolium leads to the appearance of signs of necrosis: the chromatin condenses, the oxygen consumption decreases and is uncoupled, the mitochondrial cristae are disorganized, the thiamine diphosphate-dependent dehydrogenase activities are impaired. When 10 µM thiamine are added to these cells, the basal respiration increases, the coupled respiration is restored and mitochondrial morphology is recovered within 1 h. Addition of succinate, which is oxidized via a thiamine diphosphate-independent dehydrogenase, to digitonin-permeabilized cells immediately restores a coupled respiration. Our results suggest that the slowing of the citric acid cycle is the cause of the biochemical lesion induced by severe thiamine deficiency and that part of the mitochondria remain functional. (Mol Cell Biochem 174: 121–124, 1997)     

Regional alterations of thiamine phosphate esters and of thiamine diphosphate-dependent enzymes in relation to function in experimental Wernicke's encephalopathy

Thiamine phosphate esters (thiamine monophosphate-TMP; thiamine diphosphate-TDP and thiamine triphosphate-TTP) were measured as their thiochrome derivatives by High Performance Liquid Chromatography in the brains of pyrithiamine-treated rats at various stages during the development of thiamine deficiency encephalopathy. Severe encephalopathy was accompanied by significant reductions of all three thiamine phosphate esters in brain. Neurological symptoms of thiamine deficiency appeared when brain levels of TMP and TDP fell below 15% of normal values. Activities of the TDP-dependent enzyme -ketoglutarate dehydrogenase were more severely reduced in thalamus compared to cerebral cortex, a less vulnerable brain structure. On the other hand, reductions of TTP, the non-cofactor form of thiamine, occurred to a greater extent in cerebral cortex than thalamus. Early reductions of TDP-dependent enzymes and the ensuing metabolic pertubations such as lactic acidosis impaired brain energy metabolism, and NMDA-receptor mediated excitotoxicity offer rational explanations for the selective vulnerability of brain structures such as thalamus to the deleterious effects of thiamine deficiency.     

Chronic alcoholism in rats induces a compensatory response, preserving brain thiamine diphosphate, but the brain 2-oxo acid dehydrogenases are inactivated despite unchanged coenzyme levels

Thiamine-dependent changes in alcoholic brain were studied using a rat model. Brain thiamine and its mono- and diphosphates were not reduced after 20 weeks of alcohol exposure. However, alcoholism increased both synaptosomal thiamine uptake and thiamine diphosphate synthesis in brain, pointing to mechanisms preserving thiamine diphosphate in the alcoholic brain. In spite of the unchanged level of the coenzyme thiamine diphosphate, activities of the mitochondrial 2-oxoglutarate and pyruvate dehydrogenase complexes decreased in alcoholic brain. The inactivation of pyruvate dehydrogenase complex was caused by its increased phosphorylation. The inactivation of 2-oxoglutarate dehydrogenase complex (OGDHC) correlated with a decrease in free thiols resulting from an elevation of reactive oxygen species. Abstinence from alcohol following exposure to alcohol reactivated OGDHC along with restoration of the free thiol content. However, restoration of enzyme activity occurred before normalization of reactive oxygen species levels. Hence, the redox status of cellular thiols mediates the action of oxidative stress on OGDHC in alcoholic brain. As a result, upon chronic alcohol consumption, physiological mechanisms to counteract the thiamine deficiency and silence pyruvate dehydrogenase are activated in rat brain, whereas OGDHC is inactivated due to impaired antioxidant ability.     

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.     

The upregulation of thiamine (vitamin B<Subscript>1</Subscript>) biosynthesis in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> seedlings under salt and osmotic stress conditions is mediated by abscisic acid at the early stages of this stress response

Background  

Recent reports suggest that vitamin B₁ (thiamine) participates in the processes underlying plant adaptations to certain types of abiotic and biotic stress, mainly oxidative stress. Most of the genes coding for enzymes involved in thiamine biosynthesis in Arabidopsis thaliana have been identified. In our present study, we examined the expression of thiamine biosynthetic genes, of genes encoding thiamine diphosphate-dependent enzymes and the levels of thiamine compounds during the early (sensing) and late (adaptation) responses of Arabidopsis seedlings to oxidative, salinity and osmotic stress. The possible roles of plant hormones in the regulation of the thiamine contribution to stress responses were also explored.     

Structural basis for activation of the thiamin diphosphate-dependent enzyme oxalyl-CoA decarboxylase by adenosine diphosphate

Oxalyl-coenzyme A decarboxylase is a thiamin diphosphate-dependent enzyme that plays an important role in the catabolism of the highly toxic compound oxalate. We have determined the crystal structure of the enzyme from Oxalobacter formigenes from a hemihedrally twinned crystal to 1.73 A resolution and characterized the steady-state kinetic behavior of the decarboxylase. The monomer of the tetrameric enzyme consists of three alpha/beta-type domains, commonly seen in this class of enzymes, and the thiamin diphosphate-binding site is located at the expected subunit-subunit interface between two of the domains with the cofactor bound in the conserved V-conformation. Although oxalyl-CoA decarboxylase is structurally homologous to acetohydroxyacid synthase, a molecule of ADP is bound in a region that is cognate to the FAD-binding site observed in acetohydroxyacid synthase and presumably fulfils a similar role in stabilizing the protein structure. This difference between the two enzymes may have physiological importance since oxalyl-CoA decarboxylation is an essential step in ATP generation in O. formigenes, and the decarboxylase activity is stimulated by exogenous ADP. Despite the significant degree of structural conservation between the two homologous enzymes and the similarity in catalytic mechanism to other thiamin diphosphate-dependent enzymes, the active site residues of oxalyl-CoA decarboxylase are unique. A suggestion for the reaction mechanism of the enzyme is presented.     

Non-coenzyme role of vitamin B1 in RANKL-induced osteoclastogenesis and ovariectomy induced osteoporosis

Vitamins B are co-enzymes participating in energy metabolic pathways. While some vitamins B are known affecting bone homeostasis, the effects of vitamin B1 (thiamine) on bone health remains unclear. In our study, we used cell counting kit-8, tartrate-resistant acid phosphatase stain, actin cytoskeleton stain, and pit formation assay to evaluate the effect of thiamine on osteoclast differentiation, formation, and function, respectively. Then we used dichloro-dihydro-fluorescein diacetate assay to investigate reactive oxygen species (ROS) generation and removal. Osteoporosis model by ovariectomy was established for animal experiments. We found that thiamine had inhibitory effect on osteoclast differentiation. And its inhibitory role on osteoclast differentiation is in a dose-dependent way. Mechanistically, ThDP suppresses intracellular ROS accumulation and unfolded protein response signaling during osteoclastogenesis via inhibiting Rac-Nox1/2/4 and intracellular inositol-requiring protein-1α/X-box-binding protein pathways, respectively. Osteoporotic mice treated with thiamine rich dietary showed better bone strength relative to thiamine deficient dietary. Our study explored the non-coenzyme inhibitory functions of B1 vitamin in receptor activator of nuclear factor κB ligand induced osteoclastogenesis and uncovered the significance of B1 vitamin in bone health.     

Localization of Thiamine-Binding Protein in Synaptosomes from the Rat Brain

Using preparations of synaptosomes and subsynaptosomal fractions from the rat brain, we studied the localization of thiamine-binding protein (TBP) in the subcellular structures of the neurons. In addition, we studied the distribution in synaptosomes of two types of activity typical of TBP (thiamine triphosphatase and thiamine-binding activities), as well as the effects of factors destroying the plasma membrane of synaptosomes on binding of [¹⁴C]thiamine with the latter. We found that the thiamine-associated activity of TBP was the highest in fractions of the synaptic vesicles and plasma membranes. Hydrolysis of thiamine triphosphate was also most active in these structures. Our results allow us to conclude that TBP is localized mostly in the synaptic vesicles and plasma membranes of synaptosomes.     

Induced absorption band of holotransketolase and its interpretation

It has long been known that formation of a catalytically active holotransketolase from the apoenzyme and thiamine diphosphate (ThDP) is accompanied by appearance, in both the absorption and CD spectra, of a new band. Binding and subsequent conversion of transketolase substrates bring about changes in the intensity of this band. The observation of these changes allows the investigator to monitor the coenzyme-to-apoenzyme binding and the conversion of the substrates during the transketolase reaction and thus to kinetically characterize its individual steps. As regards the new absorption band induced by ThDP binding, its nature, until recently, remained unknown. The reason for its appearance was considered to be either the formation of a charge transfer complex between ThDP and tryptophan (phenylalanine) residue or stacking interaction between the residues of aromatic amino acids. They are thought to be brought together as a result of conformational changes of the apoenzyme during its interaction with the coenzyme. However none of these hypotheses had been substantiated experimentally. According to our hypothesis, the induced absorption band is that of the imino form of ThDP resulting from three contributing features of the ThDP binding site of transketolase: the relative hydrophobicity of this site, hydrogen bonding of the N1"-atom of the ThDP aminopyrimidine ring to Glu418, and base stacking interactions between the aminopyrimidine ring of ThDP and Phe445.     

Structural determinants of specificity and catalytic mechanism in mammalian 25-kDa thiamine triphosphatase

Background

Thiamine triphosphate (ThTP) is present in most organisms and might be involved in intracellular signaling. In mammalian cells, the cytosolic ThTP level is controlled by a specific thiamine triphosphatase (ThTPase), belonging to the CYTH superfamily of proteins. CYTH proteins are present in all superkingdoms of life and act on various triphosphorylated substrates.

Methods

Using crystallography, mass spectrometry and mutational analysis, we identified the key structural determinants of the high specificity and catalytic efficiency of mammalian ThTPase.

Results

Triphosphate binding requires three conserved arginines while the catalytic mechanism relies on an unusual lysine–tyrosine dyad. By docking of the ThTP molecule in the active site, we found that Trp-53 should interact with the thiazole part of the substrate molecule, thus playing a key role in substrate recognition and specificity. Sea anemone and zebrafish CYTH proteins, which retain the corresponding Trp residue, are also specific ThTPases. Surprisingly, the whole chromosome region containing the ThTPase gene is lost in birds.

Conclusions

The specificity for ThTP is linked to a stacking interaction between the thiazole heterocycle of thiamine and a tryptophan residue. The latter likely plays a key role in the secondary acquisition of ThTPase activity in early metazoan CYTH enzymes, in the lineage leading from cnidarians to mammals.

General significance

We show that ThTPase activity is not restricted to mammals as previously thought but is an acquisition of early metazoans. This, and the identification of critically important residues, allows us to draw an evolutionary perspective of the CYTH family of proteins.     

Characterization of Thi9, a novel thiamine (Vitamin B1) transporter from Schizosaccharomyces pombe

Thiamine is an essential component of the human diet and thiamine diphosphate-dependent enzymes play an important role in carbohydrate metabolism in all living cells. Although the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe can derive thiamine from biosynthesis, both are also able to take up thiamine from external sources, leading to the down-regulation of the enzymes involved in its formation. We have isolated the S. pombe thiamine transporter Thi9 by genetic complementation of mutants defective in thiamine biosynthesis and transport. Thi9 localizes to the S. pombe cell surface and works as a high-affinity proton/thiamine symporter. The uptake of thiamine was reduced in the presence of pyrithiamine, oxythiamine, amprolium, and the thiazole part of thiamine, indicating that these compounds are substrates of Thi9. In pyrithiamine-resistant mutants, a conserved glutamate residue close to the first of the 12 transmembrane domains is exchanged by a lysine and this causes aberrant localization of the protein. Thiamine uptake is significantly increased in thiamine-deficient medium and this is associated with an increase in thi9(+) mRNA and protein levels. Upon addition of thiamine, the thi9(+) mRNA becomes undetectable within minutes, whereas the Thi9 protein appears to be stable. The protein is distantly related to transporters for amino acids, gamma-aminobutyric acid and polyamines, and not to any of the known thiamine transporters. We also found that the pyridoxine transporter Bsu1 has a marked contribution to the thiamine uptake activity of S. pombe cells.     

Quantitation of plasma thiamine,related metabolites and plasma protein oxidative damage markers in children with autism spectrum disorder and healthy controls

Abstract

Aims/hypothesis: To assess thiamine and related metabolite status by analysis of plasma and urine in autistic children and healthy controls, correlations to clinical characteristics and link to plasma protein markers of oxidative damage.

Methods: 27 children with autism (21 males and 6 females) and 21 (15 males and 6 females) age-matched healthy control children were recruited. The concentration of thiamine and related phosphorylated metabolites in plasma and urine and plasma protein content of dityrosine, N-formylkynurenine and 3-nitrotyrosine was determined.

Results: Plasma thiamine and thiamine monophosphate concentrations were similar in both study groups (median [lower–upper quartile]): autistic children – 6.60?nM (4.48–8.91) and 7.00?nM (5.51–8.55), and healthy controls – 6.82?nM (4.47–7.02) and 6.82?nM (5.84–8.91), respectively. Thiamine pyrophosphate (TPP) was decreased 24% in autistic children compared to healthy controls: 6.82?nM (5.81–8.52) versus 9.00?nM (8.41–10.71), p?<?.01. Urinary excretion of thiamine and fractional renal clearance of thiamine did not change between the groups. No correlation was observed between clinical markers and the plasma and urine thiamine concentration. Plasma protein dityrosine content was increased 88% in ASD. Other oxidative markers were unchanged.

Conclusions/interpretation: Autistic children had normal plasma and urinary thiamine levels whereas plasma TPP concentration was decreased. The latter may be linked to abnormal tissue handling and/or absorption from gut microbiota of TPP which warrants further investigation. Increased plasma protein dityrosine may reflect increased dual oxidase activity in response to change in mucosal immunity and host–microbe homeostasis.     

Biocatalytic synthesis of optically active tertiary alcohols

The enzymatic preparation of optically pure tertiary alcohols under sustainable conditions has received much attention. The conventional chemical synthesis of these valuable building blocks is still hampered by the use of harmful reagents such as heavy metal catalysts. Successful examples in biocatalysis used esterases, lipases, epoxide hydrolases, halohydrin dehalogenases, thiamine diphosphate-dependent enzymes, terpene cyclases, -acetylases, and -dehydratases. This mini-review provides an overview on recent developments in the discovery of new enzymes, their functional improvement by protein engineering, the design of chemoenzymatic routes leading to tertiary alcohols, and the discovery of entirely new biotransformations.     

Influence of donor substrate on kinetic parameters of thiamine diphosphate binding to transketolase

The two-step mechanism of interaction of thiamine diphosphate (ThDP) with transketolase (TK) has been studied: TK + ThDP <--> TK...ThDP <--> TK*-ThDP. The scheme involves the formation of inactive intermediate complex TK...ThDP followed by its transformation into catalytically active holoenzyme, TK*-ThDP. The dissociation and kinetic constants for individual stages of this process have been determined. The values of forward and backward rate constants change in the presence of the donor substrate hydroxypyruvate. This finally leads to an increase in the overall affinity of the coenzyme to TK.     

Thiamine inhibits formation of dityrosine,a specific marker of oxidative injury,in reactions catalyzed by oxoferryl forms of hemoglobin

Effects of thiamine and its derivatives on inhibition of dityrosine formation were studied in reactions catalyzed by oxoferryl forms of hemoglobin. At high thiamine concentrations, a complete inhibition of dityrosine formation was observed due to interaction of tyrosyl radicals with thiamine tricyclic and thiol forms. In neutral and alkaline media, tyrosyl radicals oxidized thiamine to thiochrome, oxodihydrothiochrome, and thiamine disulfide. In the absence of tyrosine, oxoferryl forms of hemoglobin manifested peroxidase activity towards thiamine and its phosphate esters by inducing their oxidation to disulfide compounds, thiochrome, oxodihydrothiochrome, and their phosphate esters, respectively, in neutral media. Thiamine and its phosphate esters were oxidized by both oxoferryl forms of hemoglobin, viz., +*Hb(IV=O) (compound I with an additional radical on the globin) and Hb(IV=O) (compound II). Putative mechanisms of thiamine conversions under oxidative stress and the protective role of hydrophobic thiamine metabolites are discussed.