Document Type

Thesis

Degree Name

Master of Science (MSc)

Department

Chemistry

Faculty/School

Faculty of Science

First Advisor

Dr. Geoff Horsman

Advisor Role

Thesis Supervisor

Abstract

Chemical features presented on bacterial outer surfaces play important roles in modulating surface interactions, extracellular communication, and virulence. Identifying and elucidating the biosynthesis of such features remains challenging. We have previously identified a cytidylyltransferase, PntC, which uses CTP to activate 2-aminoethylphosphonate (AEP) en route to the cell surface. The product of this transformation, CMP-AEP, resembles CDP-choline produced by the analogous LicC-catalyzed activation of phosphocholine (PCho). The activated CMP-conjugates are typically appended to a glycan or lipid in the following step. With little biochemical evidence for these technically challenging membrane-associated downstream steps in PntC/LicC-containing pathways, new tools are needed to characterize these cell surface modifications. To this end, we have engineered respective PntC and LicC enzymes from Atopobium rimae and Streptococcus pneumoniae to accept modified “Trojan horse” substrates that can be carried to the cell surface. Here we describe 2-azidoethylphosphonate (AzEP) as one such substrate that is capable of bioorthogonal reactivity through ‘click’ chemistry with alkyne partners. Because neither our PntC nor LicC enzymes activated AzEP, we engineered enzymes capable of the task. Key acidic residues were identified that were associated with substrate specificity: D105 and D192 in LicC and E114 in PntC. At all three positions, Asn variants accepted AzEP with detectable activity. The best variant, Spn-LicC D105N, turned over AzEP at a rate of 0.041 s-1, only 40-fold lower than wild-type Spn-LicC towards its cognate substrate PCho. Interestingly, the E114N substitution completely switched activity from exclusively AEP to exclusively AzEP. Finally, the viability of AzEP incorporation into cellular pathways was explored by observing that about 0.69% of AzEP entered Escherichia coli cells after 2 h of incubation with 500 µM AzEP. Overall, we have engineered an enzyme capable of accepting a clickable ‘Trojan horse’ substrate, which can be taken up by cells and therefore has the potential to be used as a biosynthetic tracking device for tagging cell wall products.

Convocation Year

2027

Convocation Season

Spring

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Available for download on Wednesday, September 29, 2027

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