Encapsulation of Transketolase into In Vitro-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. Although it has been demonstrated that encapsulation of non-native cargo proteins provides protection from protease activity, the thermal effects arising from enclosing cargo within encapsulins remain poorly understood. This study aimed to establish a methodology for loading a reporter protein into thermostable encapsulins to determine the resulting stability change of the cargo. Building on previous in vitro reassembly studies, we first investigated the effectiveness of in vitro reassembly and cargo-loading of two size classes of encapsulins Thermotoga maritima T = 1 and Myxococcus xanthus T = 3, using superfolder Green Fluorescent Protein. We show that the empty T. maritima capsid reassembles with higher yield than the M. xanthus capsid and that in vitro loading promotes the formation of the M. xanthus T = 3 capsid form over the T = 1 form, while overloading with cargo results in malformed T. maritima T = 1 encapsulins. For the stability study, a Förster resonance energy transfer (FRET)-probed industrially relevant enzyme cargo, transketolase, was then loaded into the T. maritima encapsulin. Our results show that site-specific orthogonal FRET labels can reveal changes in thermal unfolding of encapsulated cargo, suggesting that in vitro loading of transketolase into the T. maritima T = 1 encapsulin shell increases the thermal stability of the enzyme. This work supports the move toward fully harnessing structural, spatial, and functional control of in vitro assembled encapsulins with applications in cargo stabilization.

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 aromatic residue Y385 was critical to the new acceptor substrate binding, and so was explored here beyond the natural aromatic residues, to probe side chain structure and electronics effects on enzyme function and stability. A series of five variants introduced decreasing aromatic ring electron density at position 385 in the order para-aminophenylalanine (pAMF), tyrosine (Y), phenylalanine (F), para-cyanophenylalanine (pCNF) and para-nitrophenylalanine (pNTF), and simultaneously modified the hydrogen-bonding potential of the aromatic substituent from accepting to donating. The fine-tuning of residue 385 yielded variants with a 43-fold increase in specific activity for 50 mm 3-HBA and 100% increased kcat (pCNF), 290% improvement in Km (pNTF), 240% improvement in kcat /Km (pAMF) and decreased substrate inhibition relative to Y. Structural modelling suggested switching of the ring-substituted functional group, from donating to accepting, stabilised a helix-turn (D259-H261) through an intersubunit H-bond with G262, to give a 7.8 °C increase in the thermal transition mid-point, Tm , and improved packing of pAMF. This is one of the first examples in which both catalytic activity and stability are simultaneously improved via site-specific ncAA incorporation into an enzyme active site, and further demonstrates the benefits of expanding designer libraries to include ncAAs.

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, TKlow, 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, TKhigh, in the presence of Mg2+. We observed previously that partial oxidation was responsible for increased TKhigh activity, while low-activity TKlow was unmodified. In the present study, the fluorescence-based cofactor-binding assay was adapted to detect binding of the ß-hydroxypyruvate (HPA) donor substrate to wild-type transketolase and a variant, S385Y/D469T/R520Q, that is active towards aromatic aldehydes. Transketolase HPA affinity again revealed the two distinct forms of transketolase at a TKhigh:TKlow ratio that matched those observed previously via TPP binding to each variant. The HPA dissociation constant of TKlow was comparable to the substrate-inhibition dissociation constant, KiHPA, determined previously. We provide evidence that KiHPA is a convolution of binding to the low-activity TKlow-TKlow dimer, and the TKlow subunit of the partially-active TKhigh-TKlow mixed dimer, where HPA binding to the TKlow subunit of the mixed dimer results in inhibition of the active TKhigh subunit. Heat-activation of transketolase was similarly investigated and found to convert the TKlow subunit of the mixed dimer to have TKhigh-like properties, but without oxidation.

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, Mg2+. 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 [Mg2+], which explained the previous lack of consensus. A second, discrete and previously uncharacterised low-affinity TPP binding-site was also observed, and hence indicated the existence of two forms of TK with high- (TKhigh) and low-affinity (TKlow). The relative proportions of each dimer were independent of the monomer-dimer transition, as probed by analytical ultracentrifugation at various [TK]. Mass spectrometry revealed that chemical oxidation of TKlow led to the formation of TKhigh, which was 22-fold more active than TKlow. Finally, we propose a two-species model of transketolase activation that describes the interconversions between apo-/holo-TKhigh and TKlow, and the potential to significantly improve biocatalytic activity by populating only the most active form.