DigitalCommons@McMaster

Title

Characterization of Fosfomycin-Resistant MurA from Borrelia burgdorferi, Fragment-based Inhibitor Design for AroA and DAHP Synthase

Author

Shan Jiang, McMaster UniversityFollow

Date of Award

Fall 2011

Degree Type

Thesis

Degree Name

Master of Science (MSc)

Department

Chemical Biology

Supervisor

Paul J. Berti

Co-Supervisor

Giuseppe Melacini, Murray Junop

Language

English

Committee Member

Giuseppe Melacini, Murray Junop

Abstract

MurA catalyzes the first committed step of peptidoglycan biosynthesis and it is the target of the antibiotic fosfomycin. Due to a Cys-to-Asp substitution in the active site, MurAs from a number of pathogenic bacteria, including Mycobacterium tuberculosis and Borrelia burgdorferi (Lyme disease), are fosfomycin resistant. His-tagged Borrelia burgdorferi MurA (Bb_MurA) and its D116C mutant have been successfully expressed, purified and characterized. The k_cat value of wild-type Bb_MurA was 0.74 ± 0.01 s^-1. The D116C mutant’s k_cat decreased by 25-fold and was fosfomycin sensitive. The pH profiles of k_cat for both Bb_MurA and its mutant were characterized. There was little difference in pK_a1 values, but the pK_a2 value shifted from 7.4 ± 0.2 in wild-type enzyme to a value >11 in the mutant. This demonstrated that the pK_a2 of 7.4 was due to D116, and that it must be protonated for activity. Fosfomycin inactivation of Bb_MurA_H6(D116C) was time-dependent and only proceeded in the presence of UDP-GlcNAc. The dissociation constant, K_i, was 5.7 ± 0.4 µM and rate of covalent modification, k_inact, was 0.021 ± 0.003 s^-1.

DAHP synthase catalyzes the first committed step in the shikimate pathway, and its catalysis has been proposed to proceed through two oxacarbenium ion intermediates. Pyruvate oxime, glyoxylate oxime and 4-imidazolecarboxylic acid have been evaluated as inhibitors of DAHP synthase. In the presence of glycerol 3-phosphate, the fitted K_i values of pyruvate oxime and glyoxylate oxime were 7.6 (± 0.9) × 10^-5 M and 7.4 (± 1.7) × 10^-5 M, respectively. 4-Imidazolecarboxylic acid’s inhibition was cooperative, and its binding was competitive with respect to PEP, and uncompetitive with respect to E4P. Its equilibrium dissociation constant was 3.0 (± 0.2) × 10^-3 M.

Recommended Citation

Jiang, Shan, "Characterization of Fosfomycin-Resistant MurA from Borrelia burgdorferi, Fragment-based Inhibitor Design for AroA and DAHP Synthase" (2011). Open Access Dissertations and Theses. Paper 5746.
http://digitalcommons.mcmaster.ca/opendissertations/5746

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