Transketolase TKT

An impediment to a wider use of transketolases, however, was the lack of the catalyst itself, which had to be tediously purified from sources such as baker s yeast or spinach leaves [4]. With the advent of recombinant DNA technologies. 

Stereoconfiguration, and a shortened hydroxyaldehyde (glyceraldehyde 3-phosphate in the case of xylulose 5-P as donor). If hydroxypyruvate is used, carbon dioxide is evolved and leaves the reaction, thus shifting the equilibrium of the reaction to the products side. This leads to a virtually complete reaction. 

Aldehydes lacking an OH group at C2 are also transformed by transketolase, leading to a 3S configuration of the hydroxyl group in the deoxyketose product [7a, 9] albeit with a significantly lower rate than with the hydroxylated acceptors [6b, 10. In contrast to the transketolases from spinach and yeast [9, no conversion of aromatic aldehydes, e.g., benzaldehyde or hydroxybenzaldehydes, could be detected with purified E. coli transketolase [6b]. 

For synthetic purposes the E. coli transketolase has a certain advantage over the enzymes from spinach and yeast, because the conversion of a-hydroxypyru-vate with a rate of 60 U (mg of protein) [6b] is significantly higher than the rates of 2 U mg and 9 U mg reported for the spinach and yeast enzymes [9, 11], 

The gram-scale preparation of rare sugars by E. coli transketolase was demonstrated successfully for (S)-erythrulose from glycolaldehyde and hydroxypyruvate in an enzyme membrane reactor which allowed the continuous production of (S)-erythrulose with high conversion and a space-time yield of 45 g L d was reached [12]. 

---

From the many enzymes that are known to make and break C-C bonds, we first chose the two transferases, transketolase (TKT) and transaldolase (TAL), both from the Gram-negative bacterium Escherichia coli. While project B21 evolved, we learned that this microorganism holds other and so far unknown enzymes which are of interest for asymmetric syntheses. One transketolase-like enzyme, 1-deoxy-D-xylulose 5-phosphate synthase (DXS), turned out to be the first enzyme of a novel biosynthetic pathway leading to isoprenoids in bacteria, algae, and plants. The other, fructose 6-phosphate aldolase (ESA) - while similar to transaldolase - allows the direct use of the inexpensive dihydroxyacetone in aldol condensations. 

Figure 5. The biocatalytic pathway (boxed arrows) created for microbial conversion of D-glucose into cis, cw-muconate from the perspective of the biochemical pathways from which the enzymes were recruited. Conversion of D-glucose into DHS requires transketolase (tkt) from the pentose phosphate pathway and DAHP synthase (aroF, aroG, aroH)y DHQ synthase aroB and DHQ dehydratase aroD) from the common pathway of aromatic amino acid biosynthesis. Conversion of DHS into catechol requires DHS dehydratase (aroZ, enzyme A) from hydroaromatic catabolism, protocatechuate decarboxylase aroY, enzyme B), and catechol 1,2-dioxygenase (caM, enzyme C) from the benzoate branch of the p-ketoadipate pathway. (Adapted and reproduced with permission from ref. 21.)...

Transintestinal cholesterol efflux (TICE), 3446 Trans-isomers, 4066, 4069 Transketolase (TKT), 15 Transmembrane proteins, 2471 Trans-pinane, 4098 Trans-pinocarveol, 2984 Transplastomic, 2833 Transporters, extracellular, 1672... 

We have prepared several chiral and high-priced compounds on a gram scale by transketolase, DXS, or FSA (partly in collaborations with projects B25 and C). Through site-directed mutagenesis, improved biocatalysts (FSA) were obtained, or the substrate range of C-C-bonding enzymes was altered (TKT, DXS). Site-directed mutagenesis also helped to elucidate structure-function relationships at the active sites of enzymes (transketolase, DXS, PPD), when no 3D structures were available. 

Further improvement in production performance was achieved with a strain containing a tkt mutant allele (instead of the wild type) with drastically reduced enzymatic transketolase activity [180]. Transketolase is a key enzyme in the pentose phosphate pathway delivering ribulose-5-P and ribose-5-P, which are important building blocks for riboflavin biosynthesis. Presumably, the mutation impedes drainage of the pentose phosphate pathway intermediates back toward... 

---