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.

It is recognized widely that enzymes promote reactions by providing a pathway that proceeds through a transition state of lower energy. In principle, further rate enhancements could be achieved if intermediates are prevented from relaxing to their lowest energy state, and thereby reduce the barrier to the subsequent transition state. Here, we report sub-ångström-resolution crystal structures of genuine covalent reaction intermediates of transketolase. These structures reveal a pronounced out-of-plane distortion of over 20° for the covalent bond that links cofactor and substrate, and a specific elongation of the scissile substrate carbon-carbon bond (d > 1.6 Å). To achieve these distortions, the protein's conformation appears to prevent relaxation of a substrate-cofactor intermediate. The results implicate a reduced barrier to the subsequent step that is consistent with an intermediate of raised energy and leads to a more efficient overall process.

We examined the catalytic cycle of transaldolase (TAL) from Thermoplasma acidophilum by cryocrystallography and were able to structurally characterize--for the first time, to our knowledge--different genuine TAL reaction intermediates. These include the Schiff base adducts formed between the catalytic lysine and the donor ketose substrates fructose-6-phosphate and sedoheptulose-7-phosphate as well as the Michaelis complex with acceptor aldose erythrose-4-phosphate. These structural snapshots necessitate a revision of the accepted reaction mechanism with respect to functional roles of active site residues, and they further reveal fundamental insights into the general structural features of enzymatic Schiff base intermediates and the role of conformational dynamics in enzyme catalysis, substrate binding and discrimination. A nonplanar arrangement of the substituents around the Schiff base double bond was observed, suggesting that a structurally encoded reactant-state destabilization is a driving force of catalysis. Protein dynamics and the intrinsic hydrogen-bonding pattern appear to be crucial for selective recognition and binding of ketose as first substrate.

Thiamin diphosphate, the vitamin B1 coenzyme, plays critical roles in fundamental metabolic pathways that require acyl carbanion equivalents. Studies on chemical models and enzymes had suggested that these carbanions are resonance-stabilized as enamines. A crystal structure of this intermediate in pyruvate oxidase at 1.1 Å resolution now challenges this paradigm by revealing that the enamine does not accumulate. Instead, the intermediate samples between the ketone and the carbanion both interlocked in a tautomeric equilibrium. Formation of the keto tautomer is associated with a loss of aromaticity of the cofactor. The alternate confinement of electrons to neighboring atoms rather than π-conjugation seems to be of importance for the enzyme-catalyzed, redox-coupled acyl transfer to phosphate, which requires a dramatic inversion of polarity of the reacting substrate carbon in two subsequent catalytic steps. The ability to oscillate between a nucleophilic (carbanion) and an electrophilic (ketone) substrate center highlights a hitherto unrecognized versatility of the thiamin cofactor. It remains to be studied whether formation of the keto tautomer is a general feature of all thiamin enzymes, as it could provide for stable storage of the carbanion state, or whether this feature represents a specific trait of thiamin oxidases. In addition, the protonation state of the two-electron reduced flavin cofactor can be fully assigned, demonstrating the power of high-resolution cryocrystallography for elucidation of enzymatic mechanisms.crystallography | tautomerization | enzyme mechanism

Besides transketolase (TKT), a thiamin-dependent enzyme of the pentose phosphate pathway, the human genome encodes for two closely related transketolase-like proteins, which share a high sequence identity with TKT. Transketolase-like protein 1 (TKTL1) has been implicated in cancerogenesis as its cellular expression levels were reported to directly correlate with invasion efficiency of cancer cells and patient mortality. It has been proposed that TKTL1 exerts its function by catalyzing an unusual enzymatic reaction, a hypothesis that has been the subject of recent controversy. The most striking difference between TKTL1 and TKT is a deletion of 38 consecutive amino acids in the N-terminal domain of the former, which constitute part of the active site in authentic TKT. Our structural and sequence analysis suggested that TKTL1 might not possess transketolase activity. In order to test this hypothesis in the absence of a recombinant expression system for TKTL1 and resilient data on its biochemical properties, we have engineered and biochemically characterized a “pseudo-TKTL1” Δ38 deletion variant of human TKT (TKTΔ38) as a viable model of TKTL1. Although the isolated protein is properly folded under in vitro conditions, both thermal stability as well as stability of the TKT-specific homodimeric assembly are markedly reduced. Circular dichroism and NMR spectroscopic analysis further indicates that TKTΔ38 is unable to bind the thiamin cofactor in a specific manner, even at superphysiological concentrations. No transketolase activity of TKTΔ38 can be detected for conversion of physiological sugar substrates thus arguing against an intrinsically encoded enzymatic function of TKTL1 in tumor cell metabolism.

Acknowledgements 3.4.3.6. The Protonation State of DHEThDP Intermediate in EcTK-Indications for the Presence of Cofactors´ 1´-4´-Imino Tautomeric State 3.4.3.7. X-ray Structure of the DHEThDP Analouge 1,2 dihydroxyethy-3-deaza-Thiamin Diphosphate bound to hTK 3.4.4. Strain in Enzymatic Catalysis -Covalent Reaction Intermediates in TK 3.4.5. Limitations for Locating Hydrogen Positions by X-ray Crystallography -Outlook for Further Studies on Transketolases 3.4.6. Acceptor Substrate Binding in hTK and EcTK 3.4.6.1. Binding of E4P to hTK 3.4.6.2. Binding of E4P to EcTK 3.4.6.3. Binding of R5P to hTK 3.4.6.4. Usage of R5P Analogues Rib5P and 1desR5P 3.4.6.5. Binding of Rib5P to hTK 3.4.6.6. Binding of 1desR5P to hTK 3.4.6.7. Furanose Ring-Opening of R5P bound to EcTK -Proposal for an Acid/Base Mechanism 3.4.6.8. X-ray Structure of EcTK His 473 Ala in Non-Covalent Complex with R5P 3.4.6.9. Binding of Rib5P and 1desR5P to EcTK-A Structural and Thermodynamical Analysis 3.4.6.10.