Thiamin diphosphate transketolase

The activation of apo-transketolase(the enzyme protein) in erythrocyte lysate by thiamin diphosphate added in vitro has become the accepted index of thiamin nutritional status. 

The hypE proteins are 302-376 residues long and appear to consist of three domains. Domain 1 shows sequence identity to a domain from phosphoribosyl-aminoimida-zole synthetase which is involved in the fifth step in de novo purine biosynthesis and to a domain in thiamine phosphate kinase which is involved in the synthesis of the cofactor thiamine diphosphate (TDP). TDP is required by enzymes which cleave the bond adjacent to carbonyl groups, e.g. phosphoketolase, transketolase or pyruvate decarboxylase. Domain 2 also shows identity to a domain found in thiamine phosphate kinase. Domain 3 appears to be unique to the HypF proteins. 

Transketolase, which contains thiamine diphosphate, transfers C2 fragments from one sugar phosphate to another. 

The ready availability of the transketolase (TK E.C. 2.2.1.1) from E. coli within the research collaboration in G. A. Sprenger s group suggested the joint development of an improved synthesis of D-xylulose 5-phosphate 19, which was expensive but required routinely for activity measurements [27]. In vivo, transketolase catalyzes the stereospecific transfer of a hydroxyacetyl nucleophile between various sugar phosphates in the presence of a thiamine diphosphate cofactor and divalent cations, and the C2 donor component 19 offers superior kinetic constants. For synthetic purposes, the enzyme is generally attractive for its high asymmetric induction at the newly formed chiral center and high kinetic enantioselectivity for 2-hydroxyaldehydes, as well as its broad substrate tolerance for aldehyde acceptors [28]. 

Various thiamine diphosphate (ThDP)-dependent a-keto acid decarboxylases have been described as catalyzing C-C bond formation and/or cleavage [48]. Extensive work has already been conducted on transketolase (TK) and pyruvate decarboxylase (PDC) from different sources [49]. Here attention should be drawn to some concepts based on the investigation of reactions catalyzed by the enzymes... 

By 1998, X-ray structures had been determined for four thiamin diphosphate-dependent enzymes (1) a bacterial pyruvate oxidase,119120 (2) yeast and bacterial pyruvate decarboxylases,121 122c (3) transketolase,110123124 and (4) benzoylformate decarboxylase.1243 Tire reactions catalyzed by these enzymes are all quite different, as are the sequences of the proteins. However, the thiamin diphosphate is bound in a similar way in all of them. 

A number of lyases are known which, unlike the aldolases, require thiamine diphosphate (TDP) as a cofactor in the transfer of acyl anion equivalents [389-391], but proceed via enolate-type intermediates by a mechanism that resembles the classical benzoin addition. The most important representative is the transketolase (EC 2,2.1.1) [392] which stems from the oxidative pentose... 

Fig. 19. Stereo view of the active site of the transketolase from Saccaromyces cerevisiae showing the environment of the thiamine diphosphate cofactor. The magnesium ion bridging the diphosphate unit is represented as a dot...

Scheme 21. Thiamine-diphosphate-dependent decarboxylation of hydroxypyruvate by transketolase...

Transketolase removes a two-carbon fragment from ketols such as fructose 6-phosphate (alternatively xylulose 5-phosphate or sedoheptu-lose 7-phosphate) through the participation of thiamine diphosphate. Nucleophilic attack of the thiamine diphosphate anion on to the carbonyl results in an addition product which then fragments by a reverse aldol reaction, generating the chain-shortened aldose erythrose 4-phosphate, and the two-carbon carbanion unit attached to TPP (Figure 8.5) (compare the role of TPP in the decarboxylation of a-keto... 

Enantiopure, bifunctional acyloins (a-hydroxy ketones) are versatile intermediates in natural product synthesis (also see Sect. 2.3, Fig. 11). In nature, the formation of a-hydroxy ketones is efficiently catalyzed by thiamine diphosphate-dependent enzymes transketolases, decarboxylases, and other lyases, such as BALs. A great portfolio of biotransformations, especially with benzaldehyde derivatives as starting materials, were realized [204]. 

In a very imaginative piece of research Frost and coworkers have developed a plasmid-based method for synthesizing aromatic amino acids, by incorporating the genes that code for the enzymes that perform the series of conversions from D-fructose-6-phosphate to D-erythrose-4-phosphate to 3-deoxy-D-arabinoheptulosonic acid-7-phos-phate (DAHP) near each other on a plasmid that can be transformed in E. coli. The enzymes are the thiamin diphosphate-dependent enzyme transketolase in the nonoxida-tive pentose shunt and DAHP synthase. The DAHP is then converted to the cyclic dehydroquinate, a precursor to all aromatic amino acids L-Tyr, L-Phe and L-Trp165,166 (equation 27). 

Nilsson U, Meshalkma L, Lindqvist Y, Schneider G (1997) Examination of substrate binding in thiamin diphosphate-dependent transketolase by protein crystallography and site-directed mutagenesis. I Biol Chem 272 1864— 1869... 

Later studies established the coenzyme role of thiamin diphosphate in transketolase in the pentose phosphate pathway. More recent studies have shown that thiamin triphosphate acts to regulate a chloride channel in nerve tissue. 

As shown in Figure 6.4, transketolase catalyzes the transfer of a two-carbon unit from a donor ketose onto an acceptor aldose sugar. The donor ketose forms a transient intermediate with thiamin diphosphate, which then undergoes cleavage to release an aldose two carbons smaller than the ketose substrate, leaving enzyme-bound dihydroxyethyl tbiamin diphosphate. This reacts with an acceptor aldose to form a ketose two carbons larger. 

Schenk G, Duggleby RG, and Nixon PF (1998) Properties and functions of the thiamin diphosphate dependent enzyme transketolase. International Journal of Biochemistry and Cell Biology 30,1297-1318. 

Y. Lindqvist, G. Schneider, U. Ermler, and M. Sundstrom. 1992. Three-dimensional structure of transketolase, a thiamine diphosphate dependent enzyme, at 2.5 A resolution EMBO J. 11 2373-2379. (PubMed)... 

G. Schenk, R. Layfield, J.M. Candy, R.G. Duggleby, and P.F. Nixon. 1997. Molecular evolutionary analysis of the thiamine-diphosphate-dependent enzyme, transketolase J. Mol. Evol. 44 552-572. (PubMed)... 

Baines M, Davies G. The evaluation of erythrocyte thiamin diphosphate as an indicator of thiamin status in man, and its comparison with erythrocyte transketolase activity measurements. Ann Clin Biochem 1988 25 (Pt 6) 698-705. 

Deprotonation Rate of the C2-H of Thiamin Diphosphate in Transketolase from Saccharomyces cerevisiae... 

Fiedler, E., Thoeell, S., Sandalova, T., Golbik, R., Konig, S., Schneider, G. (2002), Snapshot of a key intermediate in enzymatic thiamin catalysis crystal stmeture of the a-carbanion of dihydroxyethyl)-thiamin diphosphate in the active site of transketolase from Saccharomyces cerevisiae, Proc. Nat. Acad. Sci. USA 99, 591-595. 

Konig, S., Schellenberger, A., Neee, H., Schneider, G. (1994), Specificity of coenzyme binding in thiamin diphosphate-dependent enzymes. Crystal structures of yeast transketolase in complex with analogs of thiamin diphosphate, J. Biol. Chem. 269, 10879-10882. 

A thiamin diphosphate binding fold revealed by comparison of the crystal structures of transketolase, pyruvate oxidase and pyruvate decarboxylase. Structure 1, 95-103. 

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