The Mesomeric Effect of Thiazolium on non‐Kekulé Diradicals in Pichia stipitis Transketolase

Abstract

It is theoretically plausible that thiazolium mesomerizes to congeners other than carbene in a low effective dielectric binding site; especially given the energetics and uneven electronegativity of carbene groups. However, such a phenomenon has never been reported. Nine crystal structures of transketolase obtained from Pichia stipitis (TKps) are reported with subatomic resolution, where thiazolium displays an extraordinary ring-bending effect. The bent thiazolium congeners correlate with non-Kekulé diradicals because there is no gain or loss of electrons. In conjunction with biophysical and biochemical analyses, it is concluded that ring bending is a result of tautomerization of thiazolium with its non- Kekulé diradicals, exclusively in the binding site of TKps. The chemophysical properties of these thiazolium mesomers may account for the great variety of reactivities carried out by thiamine-diphosphate-containing (ThDP) enzymes. The stability of ThDP in living systems can be regulated by the levels of substrates, and hydration and dehydration, as well as diradical-mediated oxidative degradation.

Dynamic Protonation States Underlie Carbene Formation in ThDP-Dependent Enzymes: A Theoretical Study

“…13 Among other reports, Hsu et al proposed that the activation takes place through non-Kekulédiradicals based on crystal structure data. 17 However, the structures obtained are in stark contrast to the data from other groups. Furthermore, model calculations carried out in the cofactor unequivocally show that the triplet/singlet biradical states are way too high in energy, as should be expected.…”

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

“…Given that ThDP‐hydroxyethylidene radicals were reported on several occasions, for example, pyruvate:ferredoxin oxidoreductase (PFOR), pyruvate oxidase, and α‐oxoglutarate dehydrogenase . Given also the recent identification of enzyme‐provoked non‐Kekulé diradicals of thiazolium III′ , IV , and V (Figure S2), the likelihood of a single‐electron transfer (SET) mechanism by TK is rekindled. The force that breaks the C2“−C3” bond of phosphoketose donors is another unresolved issue.…”

“…Nonetheless, the active‐site geometry and/or environment in TKps seemingly favors mesomerism of the thiazolium ring to its neutral, charged, or diradical alternatives in a dynamic manner likely through rapid thermal crossing and a captodative effect. Beyond this, desolvation in the cofactor/substrate binding site with a low effective dielectric constant is likewise cooperative for electron hopping/relay . The π radicals of thiazolium are in a position to trigger the breakage/formation of the C2“−C3” bond; thus facilitating the interconversion of phosphoketoses by virtue of this diradical mechanism.…”

“…Cloning and protein purification : The cloning and protein purification of TKps followed standard protocols . In brief, the tkt1 gene was amplified from P. stipitis CBS6054 genomic DNA by PCR and subcloned into the expression vector pET‐28a (+).…”

QM/MM Study of Human Transketolase: Thiamine Diphosphate Activation Mechanism and Complete Catalytic Cycle

“…This ThDP-activated computational structure came very close to the HuTK crystallographic structure 3MOS and was based on the deprotonated C2 ThDP, allowing no distortion of the ThDP thiazole ring. However, we noted that some high resolutions of TK structures are not compatible with the common geometry of the thiazole ring …”