Molecular Evolutionary Analysis of the Thiamine-Diphosphate-Dependent Enzyme,Transketolase

Members of the transketolase group of thiamine-diphosphate-dependent enzymes from 17 different organisms including mammals, yeast, bacteria, and plants have been used for phylogenetic reconstruction. Alignment of the amino acid and DNA sequences for 21 transketolase enzymes and one putative transketolase reveals a number of highly conserved regions and invariant residues that are of predicted importance for enzyme activity, based on the crystal structure of yeast transketolase. One particular sequence of 36 residues has some similarities to the nucleotide-binding motif and we designate it as the transketolase motif. We report further evidence that the recP protein from Streptococcus pneumoniae might be a transketolase and we list a number of invariant residues which might be involved in substrate binding. Phylogenies derived from the nucleotide and the amino acid sequences by various methods show a conventional clustering for mammalian, plant, and gram-negative bacterial transketolases. The branching order of the gram-positive bacteria could not be inferred reliably. The formaldehyde transketolase (sometimes known as dihydroxyacetone synthase) of the yeast Hansenula polymorpha appears to be orthologous to the mammalian enzymes but paralogous to the other yeast transketolases. The occurrence of more than one transketolase gene in some organisms is consistent with several gene duplications. The high degree of similarity in functionally important residues and the fact that the same kinetic mechanism is applicable to all characterized transketolase enzymes is consistent with the proposition that they are all derived from one common ancestral gene. Transketolase appears to be an ancient enzyme that has evolved slowly and might serve as a model for a molecular clock, at least within the mammalian clade. Received: 13 September 1995 / Accepted: 14 November 1996     

Western blotting assay of transketolase concentration in human hemolysates

Using a rabbit anti-human transketolase antiserum and Western blotting we can determine nanogram amounts of transketolase in human hemolysates quantitatively. Transketolase concentration in 18 apparently healthy subjects was 55.7 +/- 12.1 micrograms/g Hb (mean +/- SD). Transketolase concentration correlated positively with the enzyme activity both with and without in vitro addition of thiamin pyrophosphate. However, the former had a closer correlation (r = 0.8418, P less than 0.001) than the latter (r = 0.6703, P less than 0.01). A heavy drinker with an extremely low transketolase activity had proportionally low concentration to the activity. These results indicate that transketolase in hemolysates, whether it is holoenzyme or apoenzyme activated in vitro, has an identical specific activity among all subjects studied and that the reduced activity of transketolase in alcoholics is due to the reduced content of the enzyme protein. This method is applicable to study the dynamics and the abnormality of apotransketolase in human hemolysates.     

Inhibition of transketolase by p-hydroxyphenylpyruvate

The effect of p-hydroxyphenylpyruvate, a natural analogue of transketolase substrate, on the catalytic activity of the enzyme was investigated. p-Hydroxyphenylpyruvate proved to be a reversible and competitive inhibitor of transketolase with respect to substrate; it was also able to displace thiamine diphosphate from holotransketolase. The data suggest that p-hydroxyphenylpyruvate participates in the regulation of tyrosine biosynthesis by influencing the catalytic activity of transketolase.     

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.     

High control coefficient of transketolase in the nonoxidative pentose phosphate pathway of human erythrocytes: NMR, antibody, and computer simulation studies.

The degree of control exerted by transketolase over metabolite flux in the nonoxidative pentose phosphate pathway in human erythrocytes was investigated using transketolase antiserum to modulate the activity of that enzyme. 31P NMR enabled the simultaneous measurement of the levels of pentose phosphate pathway metabolites following incubation of hemolysates with ribose 5-phosphate. The variations in metabolic flux which occurred as the transketolase activity of hemolysate samples was altered indicated that a high degree of control was exerted by transketolase. Investigations using transaldolase-depleted hemolysates showed that transaldolase exhibits a lesser degree of control over pathway flux. Experimental data were compared with simulations generated by a computer model encompassing the reactions of the classical nonoxidative pentose phosphate pathway. The sensitivity coefficients (also called "control strengths" or "flux-control coefficients") calculated from the computer simulations were 0.74 and 0.03 for transketolase and transaldolase, respectively.     

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.     

Properties and functions of the thiamin diphosphate dependent enzyme transketolase

This review highlights recent research on the properties and functions of the enzyme transketolase, which requires thiamin diphosphate and a divalent metal ion for its activity. The transketolase-catalysed reaction is part of the pentose phosphate pathway, where transketolase appears to control the non-oxidative branch of this pathway, although the overall flux of labelled substrates remains controversial. Yeast transketolase is one of several thiamin diphosphate dependent enzymes whose three-dimensional structures have been determined. Together with mutational analysis these structural data have led to detailed understanding of thiamin diphosphate catalysed reactions. In the homodimer transketolase the two catalytic sites, where dihydroxyethyl groups are transferred from ketose donors to aldose acceptors, are formed at the interface between the two subunits, where the thiazole and pyrimidine rings of thiamin diphosphate are bound. Transketolase is ubiquitous and more than 30 full-length sequences are known. The encoded protein sequences contain two motifs of high homology; one common to all thiamin diphosphate-dependent enzymes and the other a unique transketolase motif. All characterised transketolases have similar kinetic and physical properties, but the mammalian enzymes are more selective in substrate utilisation than the nonmammalian representatives. Since products of the transketolase-catalysed reaction serve as precursors for a number of synthetic compounds this enzyme has been exploited for industrial applications. Putative mutant forms of transketolase, once believed to predispose to disease, have not stood up to scrutiny. However, a modification of transketolase is a marker for Alzheimer’s disease, and transketolase activity in erythrocytes is a measure of thiamin nutrition. The cornea contains a particularly high transketolase concentration, consistent with the proposal that pentose phosphate pathway activity has a role in the removal of light-generated radicals.     

Glycolate formation catalyzed by spinach leaf transketolase utilizing the superoxide radical

A homogeneous preparation of transketolase was obtained from spinach leaf; the specific enzyme activity was 9.5 mumolo of glyceraldehyde-3-P formed (mg of protein)-1 min-1, when xylulose-5-P and ribose-5-P were used as the donor and acceptor, respectively, of the ketol residue. Transketolase catalyzed the formation of glycolate from fructose-6-P coupled with the O2- -generating system of xanthine-xanthine oxidase. The addition of superoxide dismutase (145 units) or 1,2-dihydroxybenzene-3,5-disulfonic acid (Tiron) (5 mM), both O2- scavengers, to the reaction system inhibited glycolate formation 72 and 58%, respectively. The reacton was not inhibited by catalase. Mannitol, an .OH scavenger, and beta-carotene and 1,4-diazobicyclo[2.2.2]octane, 1O2 scavengers, showed little or no inhibitory effects. The rate of glycolate formation catalyzed by the transketolase system was measured in a coupled reaction with a continuous supply of KO2 dissolved in dimethyl sulfoxide, used as an O2- -generating system. The optimum pH of the reaction was above pH 8.5. The second-order rate constant for the reaction between transketolase and O2-, determined by the competition for O2- between nitroblue tetrazolium (NBT) and transketolase, was 1.0 X 10(6) M-1 s-1. Transketolase showed an inhibitory effect on the O2- -dependent reduction of NBT only if the reaction mixture was previously incubated with ketol donors such as fructose-6-P, xylulose-5-P, or glycolaldehyde. The results suggest the possibility that transketolase catalyzes O2- -dependent glycolate formation under increased steady-state levels of O2- in the chloroplast stroma.     

Amino acid precursors for the detection of transketolase activity in Escherichia coli auxotrophs

Probes were developed for the in vivo detection of transketolase activity by the use of a complementation assay in Escherichia coli auxotrophs They combine the d-threo ketose moiety recognised by transketolase and the side chain of leucine or methionine. These compounds were donor substrates of yeast transketolase leading to the release of the corresponding α-hydroxyaldehydes which could be converted in E. coli by a cascade of reactions into leucine or methionine required for cellular growth.     

A new method of isolation and a new form of transketolase from baker's yeast]

A new procedure for the isolation of homogeneous transketolase from baker's yeast based on the use of enzyme-specific antibodies immobilized on a insoluble matrix has been developed. The enzyme yield is 90% of its total content in the original yeast extract. The eluate from the immunocolumn was found to contain a previously unknown form of transketolase which represents an enzyme-RNA complex.     

Simultaneous assay of dihydroxyacetone synthase and transketolase in a methylotrophic yeast grown in continuous culture. A cautionary note

The methylotrophic yeast Candida boidinii CBS 5777 was grown in continuous culture under carbon limitation on glucose, glucose plus methanol, and methanol as carbon and energy sources. During adaptation from glucose to methanol there was a rapid rise in the specific activities of triokinase, fructose-1,6-bisphosphatase and dihydroxyacetone synthase, which are key enzymes of the xylulose phosphate cycle of formaldehyde fixation. The specific activity of classical transketolase fell during this adaptation. Extracts from carbon-limited C. boidinii contained an enzyme which catalysed oxidation of NADH when some preparations or ribose 5-phosphate were added, which was not a transketolase. This enzyme activity was dependent on an impurity in such ribose 5-phosphate preparations and can be confused with transketolase activity.     

In situ localization of transketolase activity in epithelial cells of different rat tissues and subcellularly in liver parenchymal cells.

Metabolic mapping of enzyme activities (enzyme histochemistry) is an important tool to understand (patho)physiological functions of enzymes. A new enzyme histochemical method has been developed to detect transketolase activity in situ in various rat tissues and its ultrastructural localization in individual cells. In situ detection of transketolase is important because this multifunctional enzyme has been related with diseases such as cancer, diabetes, Alzheimer's disease, and Wernicke-Korsakoff's syndrome. The proposed method is based on the tetrazolium salt method applied to unfixed cryostat sections in the presence of polyvinyl alcohol. The method appeared to be specific for transketolase activity when the proper control reaction is performed and showed a linear increase of the amount of final reaction product with incubation time. Transketolase activity was studied in liver, small intestine, trachea, tongue, kidney, adrenal gland, and eye. Activity was found in liver parenchyma, epithelium of small intestine, trachea, tongue, proximal tubules of kidney and cornea, and ganglion cells in medulla of adrenal gland. To demonstrate transketolase activity ultrastructurally in liver parenchymal cells, the cupper iron method was used. It was shown that transketolase activity was present in peroxisomes and at membranes of granular endoplasmic reticulum. This ultrastructural localization is similar to that of glucose-6-phosphate dehydrogenase activity, suggesting activity of the pentose phosphate pathway at these sites. It is concluded that the method developed for in situ localization of transketolase activity for light and electron microscopy is specific and allows further investigation of the role of transketolase in (proliferation of) cancer cells and other pathophysiological processes.     

A hitherto unknown transketolase-catalyzed reaction

Yeast transketolase, in addition to catalyzing the transferase reaction through utilization of two substrates--the donor substrate (ketose) and the acceptor substrate (aldose)--is also able to catalyze a one-substrate reaction with only aldose (glycolaldehyde) as substrate. The interaction of glycolaldehyde with holotransketolase results in formation of the transketolase reaction intermediate, dihydroxyethyl-thiamin diphosphate. Then the glycolaldehyde residue is transferred from dihydroxyethyl-thiamin diphosphate to free glycolaldehyde. As a result, the one-substrate transketolase reaction product, erythrulose, is formed. The specific activity of transketolase was found to be 0.23 U/mg and the apparent Km for glycolaldehyde was estimated as 140 mM.     

Overexpression of Plastid Transketolase in Tobacco Results in a Thiamine Auxotrophic Phenotype

To investigate the effect of increased plastid transketolase on photosynthetic capacity and growth, tobacco (Nicotiana tabacum) plants with increased levels of transketolase protein were produced. This was achieved using a cassette composed of a full-length Arabidopsis thaliana transketolase cDNA under the control of the cauliflower mosaic virus 35S promoter. The results revealed a major and unexpected effect of plastid transketolase overexpression as the transgenic tobacco plants exhibited a slow-growth phenotype and chlorotic phenotype. These phenotypes were complemented by germinating the seeds of transketolase-overexpressing lines in media containing either thiamine pyrophosphate or thiamine. Thiamine levels in the seeds and cotyledons were lower in transketolase-overexpressing lines than in wild-type plants. When transketolase-overexpressing plants were supplemented with thiamine or thiamine pyrophosphate throughout the life cycle, they grew normally and the seed produced from these plants generated plants that did not have a growth or chlorotic phenotype. Our results reveal the crucial importance of the level of transketolase activity to provide the precursor for synthesis of intermediates and to enable plants to produce thiamine and thiamine pyrophosphate for growth and development. The mechanism determining transketolase protein levels remains to be elucidated, but the data presented provide evidence that this may contribute to the complex regulatory mechanisms maintaining thiamine homeostasis in plants.     

Visualization of proteinase inhibitors in SDS-polyacrylamide gels

A new method is proposed for assaying transketolase activity. The method is based on determining erythrulose, the product of the transketolase reaction, by its optical activity.     

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.     

Identification and characterization of the tktB gene encoding a second transketolase in Escherichia coli K-12.

We isolated a transposon Tn10 insertion mutant of Escherichia coli K-12 which could not grow on MacConkey plates containing D-ribose. Characterization of the mutant revealed that the level of the transketolase activity was reduced to one-third of that of the wild type. The mutation was mapped at 63.5 min on the E. coli genetic map, in which the transketolase gene (tkt) had been mapped. A multicopy suppressor gene which complemented the tkt mutation was cloned on a 7.8-kb PstI fragment. The cloned gene was located at 53 min on the chromosome. Subcloning and sequencing of a 2.7-kb fragment containing the suppressor gene identified an open reading frame encoding a polypeptide of 667 amino acids with a calculated molecular weight of 72,973. Overexpression of the protein and determination of its N-terminal amino acid sequence defined unambiguously the translational start site of the gene. The deduced amino acid sequence showed similarity to sequences of transketolases from Saccharomyces cerevisiae and Rhodobacter sphaeroides. In addition, the level of the transketolase activity increased in strains carrying the gene in multicopy. Therefore, the gene encoding this transketolase was designated tktB and the gene formerly called tkt was renamed tktA. Analysis of the phenotypes of the strains containing tktA, tktB, or tktA tktB mutations indicated that tktA and tktB were responsible for major and minor activities, respectively, of transketolase in E. coli.     

Optimisation and evaluation of a generic microplate-based HPLC screen for transketolase activity

A microplate-based HPLC assay for transketolase is described for rapidly determining substrate and product concentration suitable for optimisation of biocatalytic process conditions and screening directed evolution libraries. Transketolase catalyses the enantioselective carbon-carbon bond formation of chiral keto-diol products. The assay was used to determine dissociation constants for the two cofactors required by transketolase with 5–11% error. The preparation of samples by microplate-based fermentation, cell lysis, addition of cofactor, addition of substrates was also evaluated and optimised for increased transketolase activity. The whole process enables 3-fold improved enzyme variants to be identified from a single measurement.