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15
New Applications of Transketolase: Cascade Reactions for Assay
Development

Laurence Hecquet, Wolf-Dieter Fessner, Virgil H´ elaine, and Franck Charmantray

15.1
Introduction

Transketolase (TK; EC 2.2.1.1), a thiamine diphosphate (ThDP)-dependent enzyme,
is a key enzyme in the nonoxidative branch of the pentose phosphate pathway.
TK catalyzes the stereospecific formation of a C–C bond by a reversible transfer
of the C –C ketol unit from a ketose phosphate to an aldose phosphate. The
2
1
new asymmetric center formed stereospecifically has an absolute (S) configuration
(Scheme 15.1).
TK occurs ubiquitously in all organisms. The 3D protein crystal structures of the
microbial TKs of Escherichia coli [1], Saccharomyces cerevisiae [2], Bacillus anthracis
[3], and human TK [4] have been resolved and show high structural homologies. All
these TKs are homodimers with two active sites located at the interface between the
contacting monomers. Both ThDP and bivalent cations are strictly needed for TK
activity, as is the case for all ThDP-dependent enzymes. The catalytic properties and
metabolic importance of TK support applications in biocatalysis and in medicine.
Microbial TKs have been largely used as biocatalysts for the synthesis of ketoses
and analogs [5] from nonnatural substrates such as hydroxypyruvate (HPA) as
donors and various aldehydes as acceptors. Under these conditions, the reac-
tion becomes essentially irreversible because of the release of carbon dioxide
(Scheme 15.2). TK is also enantioselective to α-hydroxyaldehyde substrates as the
acceptor component with an overwhelming preference for the (R)-configuration.
Consequently, the ketoses produced by TK-catalyzed reactions display the absolute
(3S,4R) configuration. Enzymes from E. coli and yeast are those most widely used
TKs in biocatalysis. Recently, a new TK from the thermophilic microorganism
Geobacillus stearothermophilus has attracted attention owing to its thermostable
properties, and offers interesting prospects for synthetic applications [6]. The modi-
fication of the substrate specificity of these TKs by rational or random mutagenesis
promises to broaden their application range [7]. The development of new enzymes,
and the improvement of known ones, is essential for further progress in bio-
catalysis. Such targets cause a demand for appropriate methods to quantify the

Cascade Biocatalysis: Integrating Stereoselective and Environmentally Friendly Reactions, First Edition.
Edited by Sergio Riva and Wolf-Dieter Fessner.
c 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA.