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

Abstract

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

Evidence of Diradicals Involved in the Yeast Transketolase Catalyzed Keto‐Transferring Reactions

“…A quaternary complex ( IX ) was further obtained in crystals soaked with F6P, for which F6P has been converted into a C 2 ‐keto unit dihydroxylethyl (DHE) covalently linked to ThDP (DHE‐ThDP) with a phosphoaldose E4P in close proximity to DHE in a bond association/dissociation manner (Figure F). This DHE moiety is dissimilar to hydroxylethyl‐ThDP reported by Tittmann et al., in which C2“ is of sp 3 type with a C2”−C2 bond length of 1.52 Å (Figure G). In contrast, C2“ in IX assumes an sp 2 ‐like configuration with a C2”−C2 bond length of 1.42 Å (≈1.5 bond order); this implies that the adduct is the long‐sought‐after dihydroxyethylidene (Breslow) intermediate or a DHE‐thiazolium diradical species (one is a nonbonding σ‐type radial at C2“ (sp 2 ) and the other is a π‐type radical at thiazolium).…”

“…Although the reactive C2”‐carbanion that forms a stable enamine (dihydroxyethylidene) intermediate has long been appreciated, this enamine is disfavorable to the reverse coupling reaction . Because the C2“‐hydroxyethyl‐ThDP carbanion appeared to be the dominant conformer in pyruvate oxidase, the existence of a stable ThDP enamine was thus questioned. Recently, the radical β,β‐coupling of nitroalkenes was developed by using hydroxyethylidene thiazolium as the catalyst; this highlights the radical trait of the enamine in this coupling reaction .…”

“…The resulting C2“‐carbanion thus substantiates umpolung chemistry (polarity inversion of the carbonyl electrophile to a nucleophile), which allows its coupling with a phosphoaldose acceptor in place. Although the reactive C2”‐carbanion that forms a stable enamine (dihydroxyethylidene) intermediate has long been appreciated, this enamine is disfavorable to the reverse coupling reaction . Because the C2“‐hydroxyethyl‐ThDP carbanion appeared to be the dominant conformer in pyruvate oxidase, the existence of a stable ThDP enamine was thus questioned.…”

Radical [1,3] Rearrangements of Breslow Intermediates

“…This and related heterocyclic enaminols have collectively been called "Breslow intermediates." 4 Only recently have such compounds been rigorously characterized, 5,6 and new chemistry continues to be revealed. 7 We recently reported that Breslow intermediates such as 2a derived from N-allyl benzothiazolium bromide and aromatic aldehydes could be captured in a unique Claisen rearrangement to provide 2-butenyl benzothiazoles (Scheme 2, R=H).…”

Radical [1,3] Rearrangements of Breslow Intermediates

“…3 In pyruvate decarboxylation, for example, addition of the TDP carbene to pyruvate is followed by extrusion of CO 2 to provide unstable enaminol 2 . This and related heterocyclic enaminols have collectively been called “Breslow intermediates.” 4 Only recently have such compounds been rigorously characterized, 5,6 and new chemistry continues to be revealed. 7 …”