Characterization of L,D-transpeptidase YnhG in Carbapenem Resistant Enterobacteriaceae
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Date
2022-05
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The Ohio State University
Abstract
This project is focused on analyzing and characterizing an enzyme hypothesized to
contribute to the antibiotic resistance of the pathogen Klebsiella pneumoniae. The enzymes of
interest are L,D-transpeptidases (Ldts). These proteins perform a similar task to D,Dtranspeptidases (Penicillin-Binding Proteins (PBPs)) but are sufficiently different both structurally
and in molecule-specific function that they are not recognized by standard β-lactam antibiotics.
Both Ldts and PBPs operate in the cell wall of bacteria. They work to catalyze the cross-links
between peptide stems in peptidoglycan, a network of amino acids and carbohydrates that
comprises the cell wall. Where they differ is in the specifics of the cross-link itself. PBPs recognize
a donor stem of five amino acids ending in two consecutive D-alanine's, whereas Ldts recognize
a donor stem of four amino acids ending in only one D-alanine. Generally, PBPs are the main
driving force of cell wall synthesis. However, it is hypothesized that, when under pressure from βlactam antibiotics, Ldts can take over and continue the cell wall synthesis process without
hindrance from the antibiotic. This project aimed to characterize a selected Ldt, YnhG, found in
Klebsiella pneumoniae in both its structure and function. 3D modelling via crystallography of both
apo- and holoenzyme will be utilized to study the structure of the full enzyme as well as the nature
of its active-site interactions. Kinetics assays were utilized to study the activity of YnhG. Activity
assays were performed using a chromogenic substrate nitrocefin in both solo and competition
assays. Assays using just YnhG and nitrocefin gave light to the baseline activity of the enzyme,
whereas competition assays between nitrocefin and various other select β-lactam antibiotics gave
light to the efficacy of said β-lactams against the enzyme. Both the crystallography and kinetics
assays provided critical information regarding the nature of YnhG and set the stage for novel drug
development in future research.
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Keywords
Antibiotic resistance, β-lactam, protein, column chromatography, X-ray crystallography, nitrocefin