Document Type

Thesis

Degree Name

Master of Science (MSc)

Department

Chemistry

Faculty/School

Faculty of Science

First Advisor

Geoff Horsman

Advisor Role

Principal Investigator

Abstract

Antibiotic resistance is a major global health concern that requires new therapeutic approaches. Furthermore, a lack of narrow spectrum antibiotics on the market produces unintended consequences with respect to changes in our microbial make up. Phosphonates are reduced versions of phosphates that possess a C-P bond which is more resistant to enzymatic and chemical degradation. The role of phosphonate containing macromolecules (e.g. cell surface polysaccharides) remains enigmatic, however their presence suggests that they may confer an advantage. The biosynthesis of phosphonate-containing macromolecules is unknown, but a pathway is proposed involving aLicC type cytidylyltransferase-catalyzed conjugation to a phosphonate followed by a LicD type phosphotransferase- catalyzed attachment to a macromolecule. This proposed pathway is analogous to the partially characterized phosphocholine (PC) tailoring pathway in teichoic acid biosynthesis in Streptococcus pneumoniae. In this study, the LicC homolog PngC from Atopobium rimae (Ari-PngC) was purified and its activity was compared to LicC from S. pneumoniae (Spn-LicC). Significantly, Ari-PngC preferred the phosphonate substrate 2-aminoethylphosphonate (AEP) over PC, and vice versa for Spn-LicC. Specifically, the KM and kcat values for Spn-LicC towards PC were 0.020 ±0.011 mM and 1.52 ± 0.243 s-1 respectively, yielding kcat/KM of 77.9 M-1 s-1. In contrast, the KM value for Spn-LicC towards AEP was 0.318 ± 0.126 mM and kcat of 0.722 ± 0.053 s-1, yielding kcat/km of 2.27 M-1 s-1 and revealing a 34-fold preference for PC. The opposite was seen for Ari-PngC, where AEP gave a KM value of 0.011 ± 0.001 mM , kcat of 2.72 ± 0.079 s-1 and kcat/KM of 239 M-1 s-1, while towards PC Ari-PngC yielded specificity constants of 1.67 and 0.74 when modelled by the Michaelis-Menten or substrate inhibition equation, respectively. Regardless of the correct fit, PngC clearly preferred AEP over PC. Overall, these results establish clear substrate selectivity of phosphonate versus PC tailoring pathways and set the stage for developing narrow spectrum antimicrobials.

Convocation Year

2016

Convocation Season

Fall

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