Thiamin Diphosphate Catalysis: Enzymic and Nonenzymic Covalent Intermediates

Kluger, Ronald; Tittmann, Kai

doi:10.1021/cr068444m

Cited by 250 publications

(285 citation statements)

References 244 publications

Supporting

Mentioning

272

Contrasting

Unclassified

Order By: Relevance

“…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).…”

Section: Reactions 1 Andmentioning

confidence: 99%

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

Mitschke

Parthier

Schröder-Tittmann

et al. 2010

Journal of Biological Chemistry

Self Cite

169

View full text Add to dashboard Cite

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.

show abstract

“…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).…”

Section: Reactions 1 Andmentioning

confidence: 99%

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

Mitschke

Parthier

Schröder-Tittmann

et al. 2010

Journal of Biological Chemistry

Self Cite

169

View full text Add to dashboard Cite

show abstract

“…For metal catalysis, the strong s-donor nature of NHCs makes them excellent ligands for late transition metals for asymmetric olefin metathesis [10][11][12] , hydrogenation [13][14][15][16] and conjugate additions 17,18 . Typically, an NHC-mediated organocatalytic reaction proceeds through a mechanism similar to the thiamine diphosphate mechanism, wherein a highly nucleophilic acyl-anion equivalent is the key reaction intermediate and is generated by the addition of the NHC catalyst to an aldehyde 19,20 . This umpolung species, also known as the Breslow intermediate, reacts with various electrophiles to form addition products.…”

mentioning

confidence: 99%

Asymmetric catalysis with N-heterocyclic carbenes as non-covalent chiral templates

2014

View full text Add to dashboard Cite

N-heterocyclic carbenes are a class of persistent carbenes stabilized by adjacent heteroatoms that are part of a heterocycle. They play a central role in multiple enzymatic biosynthetic reactions that involve thiamine diphosphate. Inspired by this biocatalysis machinery, N-heterocyclic carbenes have emerged as one of the most versatile classes of organocatalysts for organic reactions. However, the asymmetric synthesis of carbon-carbon bonds through a non-covalent interaction mechanism has not been previously established for chiral carbenes. Here, we report an N-heterocylic carbene-catalysed, highly enantioselective process that uses weak hydrogen bonds to relay asymmetric bias. We find that catalytic amounts of hexafluoroisopropanol are the critical proton shuttle that facilitates hydrogen transfer to provide high-reaction rates and high enantioselectivity. We demonstrate that a successful asymmetric reaction of this type can be accomplished through a rational design that balances the pKa values of the substrate, the carbene precursor and the product.

show abstract

“…1A). This step is followed by C─C bond cleavage (decarboxylation in case of α-keto acid substrates) that produces a C2α-carbanion (3a), and subsequent enzyme-controlled routes (3,4). The carbanion is potentially stabilized with the enamine contributor (3b) through delocalization into the thiazolium ring.…”

mentioning

confidence: 99%

Unexpected tautomeric equilibria of the carbanion-enamine intermediate in pyruvate oxidase highlight unrecognized chemical versatility of thiamin

Meyer

Neumann

Koers

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

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

show abstract

Thiamin Diphosphate Catalysis: Enzymic and Nonenzymic Covalent Intermediates

Cited by 250 publications

References 244 publications

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

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

Asymmetric catalysis with N-heterocyclic carbenes as non-covalent chiral templates

Unexpected tautomeric equilibria of the carbanion-enamine intermediate in pyruvate oxidase highlight unrecognized chemical versatility of thiamin

Contact Info

Product

Resources

About