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.

We recently characterised a low-activity form of E. coli transketolase, tK low , 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 high , in the presence of Mg 2+. We observed previously that partial oxidation was responsible for increased tK high activity, while lowactivity tK low was unmodified. In the present study, the fluorescence-based cofactor-binding assay was adapted to detect binding of the β-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 TK high :tK low ratio that matched those observed previously via tpp binding to each variant. the HpA dissociation constant of tK low was comparable to the substrate-inhibition dissociation constant, K i HPA , determined previously. We provide evidence that K i HPA is a convolution of binding to the low-activity tK low-tK low dimer, and the tK low subunit of the partially-active tK high-tK low mixed dimer, where HpA binding to the tK low subunit of the mixed dimer results in inhibition of the active tK high subunit. Heat-activation of transketolase was similarly investigated and found to convert the tK low subunit of the mixed dimer to have tK high-like properties, but without oxidation. Transketolase is a key enzyme of the pentose phosphate pathway (PPP), is ubiquitous in all organisms, and provides a unique link between glycolysis and the non-oxidative phase of the PPP. Transketolase is a thiamine pyrophosphate (TPP)-dependent enzyme that reversibly transfers a two-carbon ketol group from a donor substrate (usually a five-carbon ketose) to an acceptor aldehyde substrate (usually a five-carbon aldose) via a ping-pong reaction mechanism, forming a new asymmetric CC bond with high regio-and stereo-specificity. In biocatalysis, β-hydroxypyruvate (HPA) is often used as the donor substrate due to the irreversible, concomitant release of CO 2 as a by-product. Strong substrate inhibition has been observed above 25 mM HPA with an inhibition constant of around 42 mM 1,2. The cause of this substrate inhibition is currently unknown and is addressed in this study. The inactive, apo-form of transketolase is in a monomer-dimer equilibrium that is dependent on protein concentration. Upon cofactor binding, both the inactive apo-monomer and apo-dimer are converted into the catalytically active, dimeric holo-form of, until recently, seemingly structurally-identical subunits, with two active-sites per homodimer, located at the subunit interface 3,4. Each active site is comprised of one divalent cation, such as Mg 2+ , and one TPP molecule. In the S. cerevisiae transketolase apo-dimer, even after removal of free Ca 2+ , one Ca 2+ ion was bound extremely tightly to one active site and could only be removed using harsh treatment, while the second Ca 2+ ion dissociated easily. Subs...

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 k cat (pCNF), 290% improvement in K m (pNTF), 240% improvement in k cat /K m (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, T m , 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.

Correct cell fate choice is crucial in development. In post-embryonic development of the hermaphroditic Caenorhabitis elegans, distinct cell fates must be adopted in two diverse tissues. In the germline, stem cells adopt one of three possible fates: mitotic cell cycle, or gamete formation via meiosis, producing either sperm or oocytes. In the epidermis, the stem cell-like seam cells divide asymmetrically, with the daughters taking on either a proliferative (seam) or differentiated (hypodermal or neuronal) fate. We have isolated a novel conserved C. elegans tetratricopeptide repeat containing protein, TRD-1, which is essential for cell fate determination in both the germline and the developing epidermis and has homologs in other species, including humans (TTC27). We show that trd-1(RNAi) and mutant animals have fewer seam cells as a result of inappropriate differentiation towards the hypodermal fate. In the germline, trd-1 RNAi results in a strong masculinization phenotype, as well as defects in the mitosis to meiosis switch. Our data suggests that trd-1 acts downstream of tra-2 but upstream of fem-3 in the germline sex determination pathway, and exhibits a constellation of phenotypes in common with other Mog (masculinization of germline) mutants. Thus, trd-1 is a new player in both the somatic and germline cell fate determination machinery, suggestive of a novel molecular connection between the development of these two diverse tissues.

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