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Adak, Taniya

University of British Columbia

Lipopolysaccharide is a major component of the outer membrane of Gram-negative bacteria and contains an immunogenic 𝘣𝘪𝘴-phosphorylated lipid A. Cationic antimicrobial peptides and polymyxins are electrostatically attracted to the lipid A and cause membrane rupture and bacterial cell death. Therefore, many strains of Gram-negative bacteria covalently attach positively charged groups on the phosphates to decrease overall surface charge and evade antibiotic action. One such modifying group is 4-amino-4-deoxy-L-arabinose (L-Ara4N) and the biosynthesis of L-Ara4N has been studied in this thesis. One of the enzymes in the biosynthetic pathway has been hypothesized to be the deformylase, ArnD. ArnD, catalyzes the deformylation of undecaprenyl 4-formamido-4-deoxy-L-arabinosyl -α-phosphate (L-Ara4FN) to produce undecaprenyl phosphate-α-L-Ara4N, the donor substrate required for the addition of L-Ara4N to lipid A. In this work, activity of ArnD was demonstrated for the first time. This was achieved by synthesizing substrate analogs and testing for ArnD catalyzed deformylation. ArnD was shown to be a metalloenzyme and attempts were made towards the biochemical and structural characterization of the deformylase. Through experimental observations, ArnD is speculated to be a membrane-associated enzyme that partially extracts its substrate from the inner leaflet of the inner membrane. Full characterization of ArnD will allow design and testing of inhibitors, thereby paving way for new antibiotic development. In another project, mechanism-based competitive inhibitors were designed and synthesized for a few representatives from prenyltransferase class of enzymes. The inhibition of these enzymes could be useful to treat diseases like cancer and hypercholesterolemia. Enzymes that utilize allylic iv diphosphates and generate intermediates/ transition states with considerable carbocationic character were targeted. The inhibitors mimicked the transition state for substrate dissociation utilizing a planar and positively charged amidinium moiety. Contrary to our hypothesis, the inhibitors showed modest inhibition and it was concluded that not all of these enzymes evolve to stabilize a carbocation. A better inhibition strategy utilizing amidiniums could focus on enzymes that are required to evolve stabilization forces for a carbocationic intermediate.

Text

2022-12-05

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/83350

Chemistry

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/