Transketolase-catalyzed products

An enzyme membrane reactor allows continuous transketolase-catalyzed production of L-erythrulose from hydroxypyruvate and glycolaldehyde with high conversion, stable operational points, and good productivity (space-time yield) of 45 g (L d) 1, thus best overcoming transketolase deactivation by substrates (Bongs, 1997). 

Restrictions for the substrates of the transketolase-catalyzed reaction only arise from the stereochemical requirements of the enzyme. The acceptor aldehyde must be formaldehyde9,20, glycolaldehydel6,17 or a (R)-2-hydroxyaldehyde10,17. The donor ketose must exhibit a (3(7,4 R) configuration10. The enzyme selectively adds the hydroxyacetyl moiety to the Re-face of the acceptor aldehyde leading to a 3(7 configuration of the products. 

Since the equilibrium reaction mixture contains at least four products, workup can be difficult and therefore, it may be helpful to bring the reaction to completion. For example, in the transketolase-catalyzed reaction of [l-13C]D-ribosc 5-phosphate and [l-l3C]D-i/ /w-2-pen-tulose 5-phosphatc to [l,3-13C]n-a/b,o-2-heptulose 7-phosphate and D-glyceraldehyde 3-phos-... 

Transketolase catalyzes the reversible transfer of a hydroxyacetyl fragment from a ketose to an aldehyde. Because the ketose products formed by transketolase reactions are not acceptors for a consecutive transformation by the same enzyme, we have investigated the option to include a xylose (glucose) isomerase (Xyll E.C. 5.3.1.5), which has similar stereochemical specificity, for ketose to aldose equilibration (Scheme 2.2.5.13). Starting from racemic lactaldehyde 32a, the transketolase forms 5-deoxy-D-xylulose 35a, which indeed was accepted by the Xyll in situ for diastereospecific conversion into 5-deoxy-D-xylose 36a. The latter again proved to be a substrate of transketolase which completed a tandem operation to furnish 7-deoxy-sedoheptulose 37a as the sole bisadduct in 24% overall yield and in enantio- and diastereomerically pure quality [35, 36]. All four stereocenters of the resulting product are completely controlled by the enzymes during this one-pot operation. The procedure profits from the limited tolerance of the isomerase... 

Two NADPH Molecules Are Generated by the Pentose Phosphate Pathway Transaldolase and Transketolase Catalyze the Interconversion of Many Phosphorylated Sugars Production of Ribose-5-phosphate and Xylulose-5-phosphate... 

The pentose phosphate pathway also catalyzes the interconversion of three-, four-, five-, six-, and seven-carbon sugars in a series of non-oxidative reactions. All these reactions occur in the cytosol, and in plants part of the pentose phosphate pathway also participates in the formation of hexoses from CO2 in photosynthesis. Thus, D-ribulose 5-phosphate can be directly converted into D-ribose 5-phosphate by phosphopentose isomerase, or to D-xylulose 5-phosphate by phosphopentose epimerase. D-Xylulose 5-phosphate can then be combined with D-ribose 5-phosphate to give rise to sedoheptulose 7-phosphate and glyceraldehyde-3-phosphate. This reaction is a transfer of a two-carbon unit catalyzed by transketolase. Both products of this reaction can be further converted into erythrose 4-phosphate and fructose 6-phosphate. The four-carbon sugar phosphate erythrose 4-phosphate can then enter into another transketolase-catalyzed reaction with the D-xylulose 5-phosphate to form glyceraldehyde 3-phosphate and fructose 6-phosphate, both of which can finally enter glycolysis. 

In the final reaction of this type in the pathway, xylulose-5-phosphate reacts with erythrose-4-phosphate. This reaction is catalyzed by transketolase. The products of the reaction are fructose-6-phosphate and glyceraldehyde-3-phosphate (Figure 18.15, red numeral 3). 

The reactions catalyzed by transketolases are also extremely unique because of the following reason. If one sees only the starting materials and the product, the carbonyl carbon of a ketone is working as a nucleophile, which cannot happen in ordinary chemical reactions (Fig. 24). 

An example of an a-ketol formation that does not involve decarboxylation is provided by the reaction catalyzed by transketolase, an enzyme that plays an essential role in the pentose phosphate pathway and in photosynthesis (equation 21) (B-77MI11001). The mechanism of the reaction of equation (21) is similar to that of acetolactate synthesis (equation 20). The addition of (39) to the carbonyl group of (44) is followed by aldol cleavage to give a TPP-stabilized carbanion (analogous to (41)). The condensation of this carbanionic intermediate with the second substrate, followed by the elimination of (39), accounts for the observed products (B-7IMIHOO1). 

A reaction that is related to that of transketolase but is likely to function via acetyl-TDP is phosphoketolase, whose action is required in the energy metabolism of some bacteria (Eq. 14-23). A product of phosphoketolase is acetyl phosphate, whose cleavage can be coupled to synthesis of ATP. Phosphoketolase presumably catalyzes an a cleavage to the thiamin-containing enamine shown in Fig. 14-3. A possible mechanism of formation of acetyl phosphate is elimination of HzO from this enamine, tautomerization to 2-acetylthiamin, and reaction of the latter with inorganic phosphate. 

Transketolase is one of several enzymes that catalyze reactions of intermediates with a negative charge on what was initially a carbonyl carbon atom. All such enzymes require thiamine pyrophosphate (TPP) as a cofactor (chapter 10). The transketolase reaction is initiated by addition of the thiamine pyrophosphate anion to the carbonyl of a ketose phosphate, for example xylulose-5-phosphate (fig. 12.33). The adduct next undergoes an aldol-like cleavage. Carbons 1 and 2 are retained on the enzyme in the form of the glycol-aldehyde derivative of TPP. This intermediate condenses with the carbonyl of another aldolase. If the reactants are xylulose-5-phosphate and ribose-5-phosphate, the products are glyceraldehyde-3-phosphate and the seven-carbon ketose, sedoheptulose-7-phosphate (see fig. 12.33). 

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]. 

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]. 

Transaldolase catalyzes the transfer of a C3 unit. The reaction occurs via an aldol cleavage similar to that seen with aldolase there is a schiff base intermediate formed with an active site lysine. The difference between aldolase and transaldolase is in the acceptor groups in aldolase the acceptor is a proton, in transaldolase it is another sugar. This reaction yields a F-6-P, which can go to Glycolysis, and an E-4-P which reacts with Xu-5-P catalyzed by the same transketolase seen above. This second transketolase reaction yields F-6-P and Ga-3-P, both intermediates of Glycolysis and the end products of the Pentose-P pathway. 

E. In the first three reactions of the pentose phosphate pathway, glucose is converted to ribulose 5-phosphate and C02, with the production of NADPH. These reactions are not reversible. Ribose 5-phosphate and xylulose 5-phosphate may be formed from ribulose 5-phos-phate. A series of reactions catalyzed by transketolase and transaldolase produce the glycolytic intermediates fructose 6-phosphate and glyceraldehyde 3-phosphate. 

The first stage, involving the transfer of active glycolaldehyde, can be accomplished in the laboratory by use of spinach or rat-liver transketolase, and the products isolated and characterized as the barium salt and 2,7-anhydride, respectively. The second stage is catalyzed by liver or yeast transaldolase and is believed to involve the enzymic transfer of a 1,3-di-hydroxy-2-propanone residue sedoheptulose 7-phosphate and D-fructose... 

TPP is a coenzyme for transketolase, the enzyme that catalyzes the conversion of a ke-topentose (xylulose-5-phosphate) and an aldopentose (ribose-5-phosphate) into an al-dotriose (glyceraldehyde-3-phosphate) and a ketoheptose (sedoheptulose-7-phosphate). Notice that the total number of carbon atoms in the reactants and products does not change (5+5 = 3+ 7). Propose a mechanism for this reaction. 

The action of transketolase generates vicinal diols having the same stereochemistry as the products of RAMA-catalyzed condensation. The enzyme, however, has two signiHcant advantages over RAMA the reaction docs not require DHAP, and the products arc not phosphorylated. The ketose functionality can be replaced by hydroxy pyruvate, which provides a hydroxyketo equivalent after decarboxylation. No other hydroxy acid has yet been found that is accepted by transketolase. Although the enzyme is absolute in its requirement for the R configuration of the hydroxy functionality at C2 of the aldehyde, there seem to be no other stereochemical requirements. Transketolase accepts a range of aldoses as substrates, and should be a useful enzyme for carbohydrate synthesis (Table 1) (37). 

Figure 33 Biogenesis of some of the hexose-, pentose-, and cyclitol-derived components in ACAGAs from 6-IC I-D-giucose (according to Ref. 8). The labeling patterns in neomycin and vali-damycin measured by nuclear magnetic resonance (NMR) prove that all units are built up preferentially from C, (circled C atoms), C. or C, units (thick lines) rearranged by transketolase-and transaldolase-catalyzed reactions in passages through the pentosephosphate cycle. The paiiem in Cal products is hypothetical. The positions derived from the C6 of D-glucose are marked by a star.

A two-step enantioselective synthesis of 2-amino-l,3.4-butanetriol 12 was performed in continuous mode using two serial capillary microreactors with HiSg-tagged transketolase (TK) /oo-transaminase (TAM) bound to the wall via immobihzed Ni-nitrilotriacetic add (Ni-NTA) complex [78]. The TK-catalyzed conversion of hydroxypyruvate 8 and glycolaldehyde 9 to L-erythrulose 10 followed by the TAM-catalyzed amination resulted in the formation of the product This work demonstrated the implementation of a dual enzyme microreactor system for the evaluation of a de novo pathway for an enzyme-catalyzed synthesis. 

A most important clue to the nature of the steps between pentose phosphate and hexosemonophosphate, and thus to the role of the pentose phosphate pathway in photosynthesis, came from our discovery in 1953 of sedoheptulose 7-phosphate as the first product formed from pentose phosphate. The enzyme transketolase had been purified from rat liver and spinach in my laboratory and crystallized from yeast by Racker and his coworkers and the two laboratories simultaneously discovered that this enzyme contained thiamine pyrophosphate as its functional group, f Isotope studies in my laboratory showed that sedoheptulose was formed by the transfer of a C2 group ( active glycolaldehyde ) from one molecule of pentose phosphate to another, and that the reaction was fully reversible thus sedoheptulose 7-phosphate was also a Ca-donor. In addition, Racker s laboratory made the important finding that fructose 6-phosphate would also yield active glycolaldehyde, and Arturo Bonslgnore and his coworkers discovered that rat liver extracts catalyzed the rapid non-oxidative conversion of hexose phosphate to sedoheptulose phosphate. ... 

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 . 

Transketolase (EC 2.2.1.1) is involved in the oxidative pentose phosphate pathtvay in tvhich it catalyzes the reversible transfer of a hydroxyacetyl nucleophile bettveen a variety of sugar phosphates. The enzyme, tvhich requires thiamine diphosphate and divalent Mg as cofactors [248], is commercially available from baker s yeast and can be readily isolated from many natural or recombinant sources [249, 250]. The yeast enzyme has been structurally tvell characterized [251], including protein tvith a carbanion intermediate covalently bound to the cofactor [252]. Large-scale enzyme production has been investigated for the transketolase from Escherichia coli [253-255]. Immobilization vas sho vn to significantly increase stability against inactivation by aldehyde substrates [256]. The enzyme is quite tolerant to organic cosolvent, and preparative reactions have been performed continuously in a membrane reactor [255], vith potential in-situ product removal via borate complexation [257]. 

---