Publications

• His103 in yeast transketolase is required for substrate recognition and catalysis.

  Wikner C., Meshalnika L., Nilsson U., Baeckstroem S., Lindqvist Y., Schneider G.

  Crystallographic studies of thiamin-diphosphate-dependent transketolase from Saccharomyces cerevisiae suggested the invariant active-site residue H103 as a possible enzymic group binding the C1 hydroxyl group of the donor substrate and stabilizing the reaction intermediate. To test this hypothesis ... >> More

  Crystallographic studies of thiamin-diphosphate-dependent transketolase from Saccharomyces cerevisiae suggested the invariant active-site residue H103 as a possible enzymic group binding the C1 hydroxyl group of the donor substrate and stabilizing the reaction intermediate. To test this hypothesis, H103 was replaced by alanine, asparagine and phenylalanine using site-directed mutagenesis. The crystallographic analysis of the mutant transketolases verified that no structural changes occurred as a consequence of the side-chain replacements. The residual catalytic activities of the mutant enzymes were 4.3% for the H103A, 2.4% for the H103N and 0.1% for the H103F mutant transketolase. Further kinetic analysis of the H103A and H103N mutant enzymes showed that the Km values for the coenzyme were increased by about eightfold. The Km values for the acceptor substrate ribose 5-phosphate were similar to the Km value for wild-type transketolase. However, the Km value for the donor substrate, xylulose 5-phosphate is increased more than tenfold in these two mutants. Circular dichroism spectra of the mutant enzymes also indicated a weaker binding of the donor substrate and/or a less stable reaction intermediate. These observations provide further evidence in support of the proposed role for this invariant residue in recognition of the donor substrate by forming a hydrogen bond between the side chain of H103 and the C1 hydroxyl group of the sugar phosphate. The significant decrease in catalytic activity suggests that this residue also facilitates catalysis, possibly by maintaining the optimal orientation of the donor substrate and reaction intermediates. << Less

  Eur. J. Biochem. 233:750-755(1995) [PubMed] [EuropePMC]

• Effects of transketolase cofactors on its conformation and stability.

  Esakova O.A., Meshalkina L.E., Kochetov G.A.

  In studying transketolase (TK) from Saccharomyces cerevisiae, the majority of researchers use as cofactors Mg(2+) and thiamine diphosphate (ThDP) (by analogy with other ThDP-dependent enzymes), whereas the active site of native holoTK is known to contain only Ca(2+). Experiments in which Mg(2+) wa ... >> More

  In studying transketolase (TK) from Saccharomyces cerevisiae, the majority of researchers use as cofactors Mg(2+) and thiamine diphosphate (ThDP) (by analogy with other ThDP-dependent enzymes), whereas the active site of native holoTK is known to contain only Ca(2+). Experiments in which Mg(2+) was substituted for Ca(2+) demonstrated that the kinetic properties of TK varied with the bivalent cation cofactor. This led to the assumption that TK species obtained by reconstitution from apoTK and ThDP in the presence of Ca(2+) or Mg(2+), respectively, adopt different conformations. Kinetic study of the H103A mutant yeast transketolase. FEBS Letters 567, 270-274]. Analysis of far-UV circular dichroism (CD) spectra and of data, obtained using methods of thermal denaturing, differential scanning calorimetry (DSC) and tryptophan fluorescence spectroscopy, corroborated this assumption. Indeed, the ratios of secondary structure elements in the molecule of apoTK, recorded in the presence of Ca(2+) or Mg(2+), respectively, turned out to be different. The two forms of the holoenzyme, obtained by reconstitution from apoTK and ThDP in the presence of Ca(2+) or Mg(2+), respectively, also differed in stability: the holoenzyme was more stable in the presence of Ca(2+) than Mg(2+). << Less

  Life Sci 78:8-13(2005) [PubMed] [EuropePMC]

• Mutations in the transketolase-like gene TKTL1: clinical implications for neurodegenerative diseases, diabetes and cancer.

  Coy J.F., Dressler D., Wilde J., Schubert P.

  Transketolase proteins or transketolase enzyme activities have been related to neurodegenerative diseases, diabetes, and cancer. Transketolase enzyme variants and reduced transketolase enzyme activities are present in patients with the neurodegenerative disease Wernicke-Korsakoff syndrome. In Alzh ... >> More

  Transketolase proteins or transketolase enzyme activities have been related to neurodegenerative diseases, diabetes, and cancer. Transketolase enzyme variants and reduced transketolase enzyme activities are present in patients with the neurodegenerative disease Wernicke-Korsakoff syndrome. In Alzheimer's disease patients transketolase protein variants with different isoelectric points or a proteolytic cleavage leading to small transketolase protein isoforms have been identified. In diabetes mellitus patients reduced transketolase enzyme activities have been detected and the lipid-soluble thiamine derivative benfotiamine activates transketolase enzyme reactions, thereby blocking three major pathways of hyperglycemic damage and preventing diabetic retinopathy. In cancer inhibition of transketolase enzyme reactions suppresses tumor growth and metastasis. All the observed phenomena have been interpreted solely on the basis of a single transketolase gene (TKT) encoding a single transketolase enzyme. No mutations have been identified so far in TKT transketolase explaining the altered transketolase proteins or transketolase enzyme activities found in neurodegenerative diseases, diabetes and cancer. We demonstrate the presence of a second transketolase enzyme (TKTL1) in humans. During the evolution of the vertebrate genome, mutations in this transketolase gene (TKTL1) have led to tissue-specific transcripts different in size, which encode an enzymatically active transketolase protein as well as different smaller protein isoforms. The mutations within the TKTL1 gene caused a mutant transketolase enzyme with an altered substrate specificity and reaction modus. Here we characterize the TKTL1 gene and its encoded TKTL1 protein(s) and discuss the medical and clinical implications of this mutated transketolase. We furthermore postulate a novel metabolic concept for the understanding, prevention and therapy of neurodegenerative diseases, diabetes and cancer. << Less

  Clin. Lab. 51:257-273(2005) [PubMed] [EuropePMC]

• Examination of substrate binding in thiamin diphosphate-dependent transketolase by protein crystallography and site-directed mutagenesis.

  Nilsson U., Meshalkina L., Lindqvist Y., Schneider G.

  The three-dimensional structure of the quaternary complex of Saccharomyces cerevisiae transketolase, thiamin diphosphate, Ca2+, and the acceptor substrate erythrose-4-phosphate has been determined to 2.4 A resolution by protein crystallographic methods. Erythrose-4-phosphate was generated by enzym ... >> More

  The three-dimensional structure of the quaternary complex of Saccharomyces cerevisiae transketolase, thiamin diphosphate, Ca2+, and the acceptor substrate erythrose-4-phosphate has been determined to 2.4 A resolution by protein crystallographic methods. Erythrose-4-phosphate was generated by enzymatic cleavage of fructose-6-phosphate. The overall structure of the enzyme in the quaternary complex is very similar to the structure of the holoenzyme; no large conformational changes upon substrate binding were found. The substrate binds in a deep cleft between the two subunits. The phosphate group of the substrate interacts with the side chains of the conserved residues Arg359, Arg528, His469, and Ser386 at the entrance of this cleft. The aldehyde moiety of the sugar phosphate is located in the vicinity of the C-2 carbon atom of the thiazolium ring of the cofactor. The aldehyde oxygen forms hydrogen bonds to the side chains of the residues His30 and His263. One of the hydroxyl groups of the sugar phosphate forms a hydrogen bond to the side chain of Asp477. The preference of the enzyme for donor substrates with D-threo configuration at the C-3 and C-4 positions and for alpha-hydroxylated acceptor substrates can be understood from the pattern of hydrogen bonds between enzyme and substrate. Amino acid replacements by site-directed mutagenesis of residues Arg359, Arg528, and His469 at the phosphate binding site yield mutant enzymes with considerable residual catalytic activity but increased Km values for the donor and in particular acceptor substrate, consistent with a role for these residues in phosphate binding. Replacement of Asp477 by alanine results in a mutant enzyme impaired in catalytic activity and with increased Km values for donor and acceptor substrates. These findings suggest a role for this amino acid in substrate binding and catalysis. << Less

  J. Biol. Chem. 272:1864-1869(1997) [PubMed] [EuropePMC]

• Structure and properties of an engineered transketolase from maize.

  Gerhardt S., Echt S., Busch M., Freigang J., Auerbach G., Bader G., Martin W.F., Bacher A., Huber R., Fischer M.

  The gene specifying plastid transketolase (TK) of maize (Zea mays) was cloned from a cDNA library by southern blotting using a heterologous probe from sorghum (Sorghum bicolor). A recombinant fusion protein comprising thioredoxin of Escherichia coli and mature TK of maize was expressed at a high l ... >> More

  The gene specifying plastid transketolase (TK) of maize (Zea mays) was cloned from a cDNA library by southern blotting using a heterologous probe from sorghum (Sorghum bicolor). A recombinant fusion protein comprising thioredoxin of Escherichia coli and mature TK of maize was expressed at a high level in E. coli and cleaved with thrombin, affording plastid TK. The protein in complex with thiamine pyrophoshate was crystallized, and its structure was solved by molecular replacement. The enzyme is a C2 symmetric homodimer closely similar to the enzyme from yeast (Saccharomyces cerevisiae). Each subunit is folded into three domains. The two topologically equivalent active sites are located in the subunit interface region and resemble those of the yeast enzyme. << Less

  Plant Physiol. 132:1941-1949(2003) [PubMed] [EuropePMC]

• Structure and function of the transketolase from Mycobacterium tuberculosis and comparison with the human enzyme.

  Fullam E., Pojer F., Bergfors T., Jones T.A., Cole S.T.

  The transketolase (TKT) enzyme in Mycobacterium tuberculosis represents a novel drug target for tuberculosis treatment and has low homology with the orthologous human enzyme. Here, we report on the structural and kinetic characterization of the transketolase from M. tuberculosis (TBTKT), a homodim ... >> More

  The transketolase (TKT) enzyme in Mycobacterium tuberculosis represents a novel drug target for tuberculosis treatment and has low homology with the orthologous human enzyme. Here, we report on the structural and kinetic characterization of the transketolase from M. tuberculosis (TBTKT), a homodimer whose monomers each comprise 700 amino acids. We show that TBTKT catalyses the oxidation of donor sugars xylulose-5-phosphate and fructose-6-phosphate as well as the reduction of the acceptor sugar ribose-5-phosphate. An invariant residue of the TKT consensus sequence required for thiamine cofactor binding is mutated in TBTKT; yet its catalytic activities are unaffected, and the 2.5 Å resolution structure of full-length TBTKT provides an explanation for this. Key structural differences between the human and mycobacterial TKT enzymes that impact both substrate and cofactor recognition and binding were uncovered. These changes explain the kinetic differences between TBTKT and its human counterpart, and their differential inhibition by small molecules. The availability of a detailed structural model of TBTKT will enable differences between human and M. tuberculosis TKT structures to be exploited to design selective inhibitors with potential antitubercular activity. << Less

  Open Biol. 2:E10026-E10026(2012) [PubMed] [EuropePMC]

• Molecular characterization of transketolase (EC 2.2.1.1) active in the Calvin cycle of spinach chloroplasts.

  Flechner A., Dressen U., Westhoff P., Henze K., Schnarrenberger C., Martin W.

  A cDNA encoding the Calvin cycle enzyme transketolase (TKL; EC 2.2.1.1) was isolated from Sorghum bicolor via subtractive differential hybridization, and used to isolate several full-length cDNA clones for this enzyme from spinach. Functional identity of the encoded mature subunit was shown by an ... >> More

  A cDNA encoding the Calvin cycle enzyme transketolase (TKL; EC 2.2.1.1) was isolated from Sorghum bicolor via subtractive differential hybridization, and used to isolate several full-length cDNA clones for this enzyme from spinach. Functional identity of the encoded mature subunit was shown by an 8.6-fold increase of TKL activity upon induction of Escherichia coli cells that overexpress the spinach TKL subunit under the control of the bacteriophage T7 promoter. Chloroplast localization of the cloned enzyme is shown by processing of the in vitro synthesized precursor upon uptake by isolated chloroplasts. Southern blot-analysis suggests that TKL is encoded by a single gene in the spinach genome. TKL proteins of both higher-plant chloroplasts and the cytosol of non-photosynthetic eukaryotes are found to be unexpectedly similar to eubacterial homologues, suggesting a possible eubacterial origin of these nuclear genes. Chloroplast TKL is the last of the demonstrably chloroplast-localized Calvin cycle enzymes to have been cloned and thus completes the isolation of gene probes for all enzymes of the pathway in higher plants. << Less

  Plant Mol. Biol. 32:475-484(1996) [PubMed] [EuropePMC]

• Transketolase A of Escherichia coli K12. Purification and properties of the enzyme from recombinant strains.

  Sprenger G.A., Schorken U., Sprenger G., Sahm H.

  Transketolase A was purified to apparent homogeneity from recombinant Escherichia coli K12 cells carrying the homologous cloned tktA gene on a pUC19-derived plasmid. These recombinant cells exhibited a transketolase activity in crude extracts of up to 9.7 U/mg compared to < or = 0.1 U/mg in wild-t ... >> More

  Transketolase A was purified to apparent homogeneity from recombinant Escherichia coli K12 cells carrying the homologous cloned tktA gene on a pUC19-derived plasmid. These recombinant cells exhibited a transketolase activity in crude extracts of up to 9.7 U/mg compared to < or = 0.1 U/mg in wild-type cells. Transketolase A was purified from crude extracts of a recombinant strain by successive ammonium sulfate precipitations and two anion-exchange chromatography steps (Q-Sepharose FF, Fractogel EMD-DEAE column) and afforded an apparently homogeneous protein band on SDS/PAGE. The enzyme, both in its active and apoform, had a molecular mass of 145,000 Da (+/-10,000 Da), judged by gel-filtration chromatography. Subunits of 73,000 Da (+/-2000 Da) were determined on SDS/PAGE, thus, transketolase A most likely forms a homodimer. N-terminal amino acid sequencing of the protein verified the identity with the cloned gene tktA. The specific activity of the purified enzyme, determined at 30 degrees C with the substrates xylulose 5-phosphate (donor of C2 compound) and ribose 5-phosphate (acceptor) at an optimal pH (50 mM glycylglycine, pH 8.5), was 50.4 U/mg. Km values for the substrates xylulose 5-phosphate and ribose 5-phosphate were 160 microM and 1.4 mM, respectively. Km values for the other physiological substrates of transketolase A were 90 microM for erythrose 4-phosphate (best acceptor substrate), 2.1 mM for D,L-glyceraldehyde 3-phosphate, 1.1 mM for fructose 6-phosphate, and 4 mM for sedoheptulose 7-phosphate. Hydroxypyruvate served as alternative donor (Km = 18 mM). Unphosphorylated acceptor compounds were formaldehyde (Km = 31 mM), glycolaldehyde (14 mM), D,L-glyceraldehyde (10 mM) and D-erythrose (150 mM). The enzyme was competitively inhibited by D-arabinose 5-phosphate (K = 6 mM at a concentration of 2.5 mM D-arabinose 5-phosphate) or by the chelating agent EDTA. The inactive apoform of transketolase A was yielded by dialysis against buffer containing 10 mM EDTA, thus removing the cofactors thiamine diphosphate and divalent cations. The reconstitution of the apoenzyme proceded faster in the presence of manganese ions (Kd = 7 microM at 10 microM thiamine diphosphate) than with other divalent cations. << Less

  Eur. J. Biochem. 230:525-532(1995) [PubMed] [EuropePMC]

• Specificity of coenzyme binding in thiamin diphosphate-dependent enzymes. Crystal structures of yeast transketolase in complex with analogs of thiamin diphosphate.

  Konig S., Schellenberger A., Neef H., Schneider G.

  The three-dimensional structures of complexes of yeast apotransketolase with the coenzyme analogs 6'-methyl, N1'-pyridyl, and N3'-pyridyl thiamin diphosphate, respectively, were determined with protein crystallographic methods. All three coenzyme analogs bind to the enzyme in a fashion highly simi ... >> More

  The three-dimensional structures of complexes of yeast apotransketolase with the coenzyme analogs 6'-methyl, N1'-pyridyl, and N3'-pyridyl thiamin diphosphate, respectively, were determined with protein crystallographic methods. All three coenzyme analogs bind to the enzyme in a fashion highly similar to the cofactor thiamin diphosphate. Thus, either one of the hydrogen bonds of the pyrimidine ring nitrogens to the protein is sufficient for proper binding and positioning of the cofactor. The lack of catalytic activity of the N3'-pyridyl analog is not due to incorrect orientation of the pyrimidine ring, but results from the absence of the hydrogen bond between the N1' nitrogen atom and the conserved residue Glu418. The structure analysis provides further evidence for the importance of this conserved interaction for enzymatic thiamin catalysis. << Less

  J. Biol. Chem. 269:10879-10882(1994) [PubMed] [EuropePMC]

• Identification of catalytically important residues in yeast transketolase.

  Wikner C., Nilsson U., Meshalkina L., Udekwu C., Lindqvist Y., Schneider G.

  The possible roles of four histidine residues in the active site of yeast transketolase were examined by site-directed mutagenesis. Replacement of the invariant His69 with alanine yielded a mutant enzyme with 1.5% of the specific activity of the wild-type enzyme and with an increased KM for the do ... >> More

  The possible roles of four histidine residues in the active site of yeast transketolase were examined by site-directed mutagenesis. Replacement of the invariant His69 with alanine yielded a mutant enzyme with 1.5% of the specific activity of the wild-type enzyme and with an increased KM for the donor. This residue is located at the bottom of the substrate cleft close to the C1 hydroxyl group of the donor substrate, and the side chain of His69 might be required for recognition of this hydroxyl group and possibly for maintenance of the proper orientation of the reaction intermediate, (alpha, beta-dihydroxyethyl)thiamin diphosphate. Amino acid replacements of His481 by alanine, serine, and glutamine resulted in mutant enzymes with significantly increased KM values for the donor substrate and specific activities of 4.4%, 1.9%, and 5.5% of the wild-type enzyme. The kinetic data suggest that this residue, although close to the C2 carbonyl oxygen of the substrate, is not absolutely required for stabilization of the negative charge that develops at this oxygen in the transition state. This points toward the 4'-NH2 group of the pyrimidine ring of thiamin diphosphate as the major source of charge stabilization. Mutations at positions His30 and His263 result in mutant enzymes severely impaired in catalytic activity (1.5% and less of the activity of wild-type transketolase). The KM value for the donor substrate was increased for the His30Ala mutant but remained unchanged in the His263Ala enzyme. The side chains of both residues interact with the C3 hydroxyl group of the donor substrate, and the results indicate that the two residues act in concert during proton abstraction of the C3 hydroxyl proton during catalysis. << Less

  Biochemistry 36:15643-15649(1997) [PubMed] [EuropePMC]

• Properties and functions of the thiamin diphosphate dependent enzyme transketolase.

  Schenk G., Duggleby R.G., Nixon P.F.

  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 th ... >> More

  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. << Less

  Int J Biochem Cell Biol 30:1297-1318(1998) [PubMed] [EuropePMC]

• Mutations in TKT are the cause of a syndrome including short stature, developmental delay, and congenital heart defects.

  Boyle L., Wamelink M.M., Salomons G.S., Roos B., Pop A., Dauber A., Hwa V., Andrew M., Douglas J., Feingold M., Kramer N., Saitta S., Retterer K., Cho M.T., Begtrup A., Monaghan K.G., Wynn J., Chung W.K.

  Whole-exome sequencing (WES) is increasingly being utilized to diagnose individuals with undiagnosed disorders. Developmental delay and short stature are common clinical indications for WES. We performed WES in three families, using proband-parent trios and two additional affected siblings. We ide ... >> More

  Whole-exome sequencing (WES) is increasingly being utilized to diagnose individuals with undiagnosed disorders. Developmental delay and short stature are common clinical indications for WES. We performed WES in three families, using proband-parent trios and two additional affected siblings. We identified a syndrome due to an autosomal-recessively inherited deficiency of transketolase, encoded by TKT, on chromosome 3p21. Our series includes three families with a total of five affected individuals, ranging in age from 4 to 25 years. Two families of Ashkenazi Jewish ancestry were homozygous for an 18 base pair in-frame insertion in TKT. The third family was compound heterozygous for nonsense and missense variants in TKT. All affected individuals had short stature and were developmentally delayed. Congenital heart defects were noted in four of the five affected individuals, and there was a history of chronic diarrhea and cataracts in the older individuals with the homozygous 18 base pair insertion. Enzymatic testing confirmed significantly reduced transketolase activity. Elevated urinary excretion of erythritol, arabitol, ribitol, and pent(ul)ose-5-phosphates was detected, as well as elevated amounts of erythritol, arabitol, and ribitol in the plasma of affected individuals. Transketolase deficiency reduces NADPH synthesis and nucleic acid synthesis and cell division and could explain the problems with growth. NADPH is also critical for maintaining cerebral glutathione, which might contribute to the neurodevelopmental delays. Transketolase deficiency is one of a growing list of inborn errors of metabolism in the non-oxidative part of the pentose phosphate pathway. << Less

  Am. J. Hum. Genet. 98:1235-1242(2016) [PubMed] [EuropePMC]

• Strain and near attack conformers in enzymic thiamin catalysis: X-ray crystallographic snapshots of bacterial transketolase in covalent complex with donor ketoses xylulose 5-phosphate and fructose 6-phosphate, and in noncovalent complex with acceptor aldose ribose 5-phosphate.

  Asztalos P., Parthier C., Golbik R., Kleinschmidt M., Hubner G., Weiss M.S., Friedemann R., Wille G., Tittmann K.

  Transketolase is a prominent thiamin diphosphate-dependent enzyme in sugar metabolism that catalyzes the reversible transfer of a 2-carbon dihydroxyethyl fragment between a donor ketose and an acceptor aldose. The X-ray structures of transketolase from E. coli in a covalent complex with donor keto ... >> More

  Transketolase is a prominent thiamin diphosphate-dependent enzyme in sugar metabolism that catalyzes the reversible transfer of a 2-carbon dihydroxyethyl fragment between a donor ketose and an acceptor aldose. The X-ray structures of transketolase from E. coli in a covalent complex with donor ketoses d-xylulose 5-phosphate (X5P) and d-fructose 6-phosphate (F6P) at 1.47 A and 1.65 A resolution reveal significant strain in the tetrahedral cofactor-sugar adducts with a 25-30 degrees out-of-plane distortion of the C2-Calpha bond connecting the substrates' carbonyl with the C2 of the cofactor's thiazolium part. Both intermediates adopt very similar extended conformations in the active site with a perpendicular orientation of the scissile C2-C3 sugar bond relative to the thiazolium ring. The sugar-derived hydroxyl groups of the intermediates form conserved hydrogen bonds with one Asp side chain, with a cluster of His residues and with the N4' of the aminopyrimidine ring of the cofactor. The phosphate moiety is held in place by electrostatic and hydrogen-bonding interactions with Arg, His, and Ser side chains. With the exception of the thiazolium part of the cofactor, no structural changes are observable during intermediate formation indicating that the active site is poised for catalysis. DFT calculations on both X5P-thiamin and X5P-thiazolium models demonstrate that an out-of-plane distortion of the C2-Calpha bond is energetically more favorable than a coplanar bond. The X-ray structure with the acceptor aldose d-ribose 5-phosphate (R5P) noncovalently bound in the active site suggests that the sugar is present in multiple forms: in a strained ring-closed beta-d-furanose form in C2-exo conformation as well as in an extended acyclic aldehyde form, with the reactive C1 aldo function held close to Calpha of the presumably planar carbanion/enamine intermediate. The latter form of R5P may be viewed as a near attack conformation. The R5P binding site overlaps with those of the leaving group moieties of the covalent donor-cofactor adducts, demonstrating that R5P directly competes with the donor-derived products glyceraldehyde 3-phosphate and erythrose 4-phosphate, which are substrates of the reverse reaction, for the same docking site at the active site and reaction with the DHEThDP enamine. << Less

  Biochemistry 46:12037-12052(2007) [PubMed] [EuropePMC]