The Crystal Structure of Human Transketolase and New Insights into Its Mode of Action

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. Transketolase (TKT 3 ; EC 2.2.1.1) is a ubiquitous enzyme in cellular carbon metabolism and requires thiamine diphosphate (ThDP), the biologically active derivative of vitamin B1, and Ca 2ϩ ions as cofactors for enzymatic activity (1, 2). TKT catalyzes the reversible transfer of two-carbon (1,2-dihydroxyethyl) units from ketose phosphates to the C1 position of aldose phosphates and thus provides, together with the Schiff base-forming transaldolase, a reversible link between glycolysis and the pentose phosphate pathway. This shunt permits cells a flexible adaptation to different metabolic needs as the pentose phosphate pathway supplies intermediates for other metabolic pathways; generates precursors for biosynthesis of nucleotides, aromatic amino acids, and vitamins; and further produces NADPH for sustaining the glutathione level and for reductive biosynthetic pathways of, for example, cholesterol and fatty acids.TKT acts on different ketose phosphate (donor) and aldose phosphate (acceptor) substrates of variable carbon chain length (3-7 carbons) in two major, essentially reversible reactions. REACTIONS 1 AND 2A simplified reaction scheme for the TKT-catalyzed conversion of substrates X5P and R5P into products S7P and G3P is shown in Fig. 1. The reaction cycle can be subdivided into a donor half-reaction (donor ligation and cleavage) and an acceptor half-reaction (acceptor ligation and product liberation) (3).After formation of the reactive ylide form of ThDP, the C2 carbanion of ThDP attacks the carbonyl of donor X5P in a nucleophilic manner to yield the covalent donor-ThDP adduct X5P-ThDP (step 1). Ionization of C3-OH and cleavage of the scissile C2-C3 bond of X5P-ThDP results in the formation of product G3P and of the 1,2-dihydroxylethyl-ThDP (DHEThDP) carbanion/enamine intermediate (step 2). This intermediate may then react with either G3P (reverse reaction of step 2) or R5P in competing equilibria. In the latter case, C2␣ of DHE-ThDP ligates to C1 of R5P (in the acyclic form), yielding * This work was supported by a grant from the "Fonds der Chemischen Industrie" (stipend to S. L.) and the Deutsche Forschungsgemeinschaft-funded Gö ttingen Graduate School for Neurosciences and Molecular Biosciences (to K. T.

Sub-ångström-resolution crystallography reveals physical distortions that enhance reactivity of a covalent enzymatic intermediate

“…According to refinement statistics, 75-80% of the active sites contain the covalently bonded intermediate in crystallo, and the remaining ones contain unreacted cofactor. This agrees with results from 1 H NMR spectroscopic analyses of related species in solution 9,11 . Coordinates for all intermediate atoms were obtained from geometrically unrestrained refinement, which is only feasible at ultrahigh resolution.…”

“…Human TK that contains a C-terminal His 6 -tag was expressed in Escherichia coli BL21Cþ by high cell-density fermentation, purified by immobilized metal-affinity chromatography and gel filtration, and finally crystallized as described recently 9 . Prior to X-ray data collection, single crystals were soaked for one minute in cryoprotectant that contained 20% (w/v) PEG-6000, 30% (v/v) ethylene glycol, 5 mM ThDP, 10 mM CaCl 2 in 50 mM glycylglycine, pH 7.9 supplemented with 100 mM X5P, F6P or S7P.…”

“…Data were processed and scaled with XDS software 29 . The previously determined 1.75 Å structure of human TK (Protein Data Bank (PDB) code 3MOS) 9 was used as a starting model for initial rigid-body refinements. Further iterative cycles of model building and refinement were carried out using COOT 30 , PHENIX 31 and finally SHELXL-2012 12 until R free and R cryst converged.…”

“…TK catalyses the reversible transfer of two carbon units between sugar phosphates as part of the pentose phosphate pathway, utilizing the cofactor thiamin diphosphate (ThDP) for catalytic activity 9,10 . In the physiological context, TK transfers a two-carbon fragment from ketose D-xylulose-5-phosphate (X5P) to the alternative aldose acceptors D-erythrose-4-phosphate (E4P) and D-ribose-5-phosphate (R5P) to yield products D-fructose-6-phosphate (F6P) or D-sedoheptulose-7-phosphate (S7P) plus D-glyceraldehyde-3-phosphate ( Fig.…”

An efficient amperometric transketolase assay: Towards inhibitor screening

“…The 3D protein crystal structures of the microbial TKs from Escherichia coli (Littlechild et al, 1995), Saccharomyces cerevisiae (Sundstrom et al, 1993), Bacillus anthracis (Maltseva et al, 2009) and human TK (Mitschke et al, 2010) 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.…”