Mutagenic Analysis of ArnD, an Enzyme Essential for Polymyxin Resistance

Wilcox-Snyder, Blake Marleau

Undergraduate Honors Thesis

Mutagenic Analysis of ArnD, an Enzyme Essential for Polymyxin Resistance Public Deposited

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Citeable URL: https://scholar.colorado.edu/concern/undergraduate_honors_theses/m039k533p

Abstract

Cystic Fibrosis (CF) is the most frequently inherited complication among Caucasians today, affecting 30,000 people in the U.S. alone. The major cause of mortality within CF patients is the development of chronic persistent infections by the opportunistic pathogen Pseudomonas aeruginosa. Typical antibacterial defense mechanisms used within healthy individuals such as Cationic Antimicrobial Peptides (CAMPs) are inhibited within CF patients. The inhibition is mediated by modification of the bacterial surface in the specific environment of the CF lung, namely: the covalent addition of Ara4NH4+ to Lipid A. This unique Lipid A modification utilized by P. aeruginosa within CF patients has been proven to establish a resistance against both CAMPs and last resort antibiotics such as Polymyxins. Abolishing this Lipid A modification would help control the pulmonary infections and thus holds significant promise as a means of extending the life expectancy and improving the quality of life of CF patients. The enzymes responsible for mediating a resistance against CAMPs are encoded by the seven-enzyme operon ArnBCADTEF. Here, I show that ArnD, an essential enzyme within the biosynthetic pathway, is capable of converting Undecaprenyl Phosphate-L-Ara4N-formyl to a novel species, which I propose is the deformylated intermediate Undecaprenyl Phosphate-L-Ara4N. I further show that the catalytic activity of ArnD is dependent on residues D43 and H233 as mutation of these residues dramatically reduces ArnD activity. This is consistent with ArnD catalyzing the deformylation reaction by a mechanism analogous to that of polysaccharide deacetylases, a family of metal dependent hydrolases. This would establish ArnD as the essential deformylase within the reaction pathway that leads to Lipid A Ara4NH4+ biosynthesis and bacterial resistance to CAMPs and clinical antibiotics of the same family such as Polymyxin B and Colistin.

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