Encapsulation of Transketolase into -Assembled Protein Nanocompartments Improves Thermal Stability.

Protein compartments offer definitive structures with a large potential design space that are of particular interest for green chemistry and therapeutic applications. One family of protein compartments, encapsulins, are simple prokaryotic nanocompartments that self-assemble from a single monomer into selectively permeable cages of between 18 and 42 nm. Over the past decade, encapsulins have been developed for a diverse application portfolio utilizing their defined cargo loading mechanisms and repetitive surface display.

Fine-tuning the activity and stability of an evolved enzyme active-site through noncanonical amino-acids.

Site-specific saturation mutagenesis within enzyme active sites can radically alter reaction specificity, though often with a trade-off in stability. Extending saturation mutagenesis with a range of noncanonical amino acids (ncAA) potentially increases the ability to improve activity and stability simultaneously. Previously, an Escherichia coli transketolase variant (S385Y/D469T/R520Q) was evolved to accept aromatic aldehydes not converted by wild-type.

The Two-Species Model of transketolase explains donor substrate-binding, inhibition and heat-activation.

We recently characterised a low-activity form of E. coli transketolase, TK, which also binds the cofactor thiamine pyrophosphate (TPP) with an affinity up to two-orders of magnitude lower than the previously known high TPP-affinity and high-activity form, TK, in the presence of Mg. We observed previously that partial oxidation was responsible for increased TK activity, while low-activity TK was unmodified.

Novel insights into transketolase activation by cofactor binding identifies two native species subpopulations.

Transketolase (TK) cofactor binding has been studied extensively over many years, yet certain mysteries remain, such as a lack of consensus on the cooperativity of thiamine pyrophosphate (TPP) binding into the two active sites, in the presence and absence of the divalent cation, Mg. Using a novel fluorescence-based assay, we determined directly the dissociation constants and cooperativity of TPP binding and provide the first comprehensive study over a broad range of cofactor concentrations. We confirmed the high-affinity dissociation constants and revealed a dependence of both the affinity and cooperativity of binding on [Mg], which explained the previous lack of consensus.