Abstract
The crystal structure of human transketolase (TKT), a thiamine diphosphate (ThDP) and Ca(2+)-dependent enzyme that catalyzes the interketol transfer between ketoses and aldoses as part of the pentose phosphate pathway, has been determined to 1.75 Å resolution. The recombinantly produced protein crystallized in space group C2 containing one monomer in the asymmetric unit. Two monomers form the homodimeric biological assembly with two identical active sites at the dimer interface. Although the protomer exhibits the typical three (α/β)-domain structure and topology reported for TKTs from other species, structural differences are observed for several loop regions and the linker that connects the PP and Pyr domain. The cofactor and substrate binding sites of human TKT bear high resemblance to those of other TKTs but also feature unique properties, including two lysines and a serine that interact with the β-phosphate of ThDP. Furthermore, Gln(189) spans over the thiazolium moiety of ThDP and replaces an isoleucine found in most non-mammalian TKTs. The side chain of Gln(428) forms a hydrogen bond with the 4'-amino group of ThDP and replaces a histidine that is invariant in all non-mammalian TKTs. All other amino acids involved in substrate binding and catalysis are strictly conserved. Besides a steady-state kinetic analysis, microscopic equilibria of the donor half-reaction were characterized by an NMR-based intermediate analysis. These studies reveal that formation of the central 1,2-dihydroxyethyl-ThDP carbanion-enamine intermediate is thermodynamically favored with increasing carbon chain length of the donor ketose substrate. Based on the structure of human transketolase and sequence alignments, putative functional properties of the related transketolase-like proteins TKTL1 and -2 are discussed in light of recent findings suggesting that TKTL1 plays a role in cancerogenesis.
Highlights
TKT acts on different ketose phosphate and aldose phosphate substrates of variable carbon chain length (3–7 carbons) in two major, essentially reversible reactions
Sequence analysis further revealed that ϳ50 amino acids are invariant across all species, including many residues shown to be involved in cofactor and substrate binding, such as a cluster of histidine and arginine residues
Previous studies had revealed that a heterologous expression of human TKT in bacterial hosts resulted in poor yields due to very modest expression levels, which allowed a functional characterization but did not provide sufficient amounts for crystallization and eventual x-ray structural analysis (40)
Summary
Yeast, and plant enzymes comprise about ϳ45–50% identical amino acids, mammalian TKTs share less identity with TKTs from other organisms (4). In view of the central role of TKT in normal and different disease states, human TKT appears to be a promising drug target. In this context, it was previously demonstrated that the activity of human TKT could be effectively decreased both in vivo and in vitro by the addition of inactive cofactor analogues (24 –27). A marked difference between TKT and TKTL1 is a deletion of 38 amino acids in the N-terminal PP domain (residues 76 –113 in TKT), including 4 residues (Tyr, Gly, His110, and Pro111) that are totally invariant among all transketolase sequences (4). We hypothesize about the functional relationship of TKT and the proteins TKTL1 and TKTL2
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