Transketolase catalysis

Figure 10.39 Synthesis of a novel N-hydroxypyrrolidine and a fluorogenic screening substrate for transaldolases based on stereospecific transketolase catalysis.

The reactive open-chain substrate 29 with the natural D-threo configuration was prepared along a chemoenzymatic route by making use of the common constitutional and stereochemical relationship which substrates of transaldolase share with those of transketolase. Thus, the R-configured 2-hydroxyaldehyde 28 was chain-extended under transketolase catalysis in the presence of 20 as ketol donor to yield the desired aldol. By this approach, several transaldolases could indeed be shown to display different levels of kinetic stereoselectivity. 

On the basis of the crystal structure of a Bacillus stearothermophilus pyruvate dehydrogenase subcomplex formed between the heterotetrameric El and the peripheral subunit binding domain of E2 with an evident stmctural dissymmetry of the two active sites, a direct active center communication via an acidic proton tunnel has been proposed (Frank et ak, 2004). According to this, one active site is in a closed state with an activated cofactor even before a substrate molecule is engaged, whereas the activation of the second active site is coupled to decarboxylation in the first site. Our own kinetic NMR studies on human PDH El (unpublished) support the model suggested, but similar studies on related thiamin enzymes, such as pyruvate decarboxylase, transketolase or pyruvate oxidase reveal that half-of-the-sites reactivity is a unique feature of ketoacid dehydrogenases. In line with this. X-ray crystallography studies on intermediates in transketolase catalysis indicated an active site occupancy close to unity in both active sites (Fiedler et al., 2002 and G. Schneider, personal communication). 

The complexation of a resorcinol-dodecanal cyclotetramer as achiral host with sugars as guest compounds has been reported. Evidence has been presented of a biological radical deoxygenation step in the biosynthesis of ascarylose (3,6-dideoxy-L-aroh /io-hexose). The role of transketolase catalysis in the conversion of D-glucose to aromatic amino acids has been investigated with a view to its application to a commercial process. ... 

Synthesis of a novel /V-hydroxypyrrolidine on the basis of transketolase catalysis. 

Ketoses have been prepared using transketolase catalysis. The enzyme, isolated from Saccharomyces cerevisiae (baxer s yeast) or from spinacb leaf was investigated for substrate specificity and it ivas shown that it was not necessary for the ketose to be phosphory-lated. The general biosynthetic condensation shown in Scheme 5 was used in the preparation of L-erythrulose from glycolaldehyde, D-xylulose from D- or D,L-glyceraldehyde, and 3-deoxy-U-xylulose from... 

A number of mechanistically distinct enzymes can likewise be employed for the synthesis of product structures identical to those accessible from aldolase catalysis. Such alternative cofactor-dependent enzymes (e.g. transketolase) are emerging as useful catalysts in organic synthesis. As these operations often extend and/or... 

The transketolase (TK EC 2.2.1.1) catalyzes the reversible transfer of a hydroxy-acetyl fragment from a ketose to an aldehyde [42]. A notable feature for applications in asymmetric synthesis is that it only accepts the o-enantiomer of 2-hydroxyaldehydes with effective kinetic resolution [117, 118] and adds the nucleophile stereospecifically to the re-face of the acceptor. In effect, this allows to control the stereochemistry of two adjacent stereogenic centers in the generation of (3S,4R)-configurated ketoses by starting from racemic aldehydes thus this provides products stereochemically equivalent to those obtained by FruA catalysis. The natural donor component can be replaced by hydroxy-pyruvate from which the reactive intermediate is formed by a spontaneous decarboxylation, which for preparative purposes renders the overall addition to aldehydic substrates essentially irreversible [42]. 

In these reactions, the C2-atom of ThDP must be deprotonated to allo v this atom to attack the carbonyl carbon of the different substrates. In all ThDP-dependent enzymes this nucleophilic attack of the deprotonated C2-atom of the coenzyme on the substrates results in the formation of a covalent adduct at the C2-atom of the thiazolium ring of the cofactor (Ila and Ilb in Scheme 16.1). This reaction requires protonation of the carbonyl oxygen of the substrate and sterical orientation of the substituents. In the next step during catalysis either CO2, as in the case of decarboxylating enzymes, or an aldo sugar, as in the case of transketo-lase, is eliminated, accompanied by the formation of an a-carbanion/enamine intermediate (Ilia and Illb in Scheme 16.1). Dependent on the enzyme this intermediate reacts either by elimination of an aldehyde, such as in pyruvate decarboxylase, or with a second substrate, such as in transketolase and acetohydroxyacid synthase. In these reaction steps proton transfer reactions are involved. Furthermore, the a-carbanion/enamine intermediate (Ilia in Scheme 16.1) can be oxidized in enzymes containing a second cofactor, such as in the a-ketoacid dehydrogenases and pyruvate oxidases. In principal, this oxidation reaction corresponds to a hydride transfer reaction. 

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. 

Wikner, C., Meshalkina, L., Nilsson, U., Nikkola, M., Lindqvist, Y., Sundstrom, M., Schneider, G. (1994), Analysis of an invariant cofactor-protein interaction in thiamin diphosphate-dependent enzymes by site-directed mutagenesis. Glutamic acid 418 in transketolase is essential for catalysis,/. Biol. Chem. 269, 32144-32150. 

Schneider, G., and Lindqvist, Y., 1998. Crystallography and mutagenesis of transketolase mechanistic implications for enzymatic thiamin catalysis. Biochimica et Biophysica Acta. 1385 387-398. 

In addition to the preparatively useful aldolases, several mechanistically distinct enzymes can be employed for synthesis of product structures identical w ith those accessible from aldolase catalysis. Such alternative enzymes (e.g. transketolase), w hich are actually categorized as transferases but also catalyze aldol-related additions w ith the aid of cofactors (Eigure 5.4) such as pyridoxal 5-phosphate (PEP), thiamine pyrophosphate (TPP), or tetra-hydrofolate (THE), are emerging as useful catalysts in organic synthesis. Because these operations often extend and/or complement the synthetic strategies open to aldolases, a selection of such enzymes and examples of their synthetic utility are included also in this overview . 

TK plays a vital role in sugar metabolism. In vivo, transketolase acts as transferase that catalyzes the transfer of an a-ketol group from a ketose phosphate to an aldose phosphate by ThDP catalysis (Scheme 28.29). 

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