In vitro Biochemical Studies of the Decarboxylase Domain of ArnA from Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is an opportunistic, Gram‐negative pathogen that is resistant to many classes of antibiotics. One pathway leading to resistance to cationic antimicrobial peptides like colistin is upregulation of the arnBCADTEF operon. With the biosynthetic enzymes encoded in this pathway, P. aeruginosa modifies its outer membrane to decrease its net negative charge. This neutralization of charge lessens the driving force for electrostatic interactions with positively charged antibiotics such as colistin, hindering their entry into the cell. The first committed step of this pathway is catalyzed by the decarboxylase domain of ArnA in which UDP‐glucuronic acid undergoes a redox reaction with NAD+. Due to its role in the arnB pathway of antibiotic resistance, we seek to characterize the kinetics of ArnA using in vitro biochemical studies. To do this we measured the change in absorbance at 340 nm allowing us to calculate the reduction of NAD+ to NADH. These data were analyzed to produce the Michaelis‐Menten kinetics of ArnA from P. aeruginosa. Elucidating the kinetics of ArnA will enable analysis of small molecule inhibitors for changes in the activity of this enzyme. This knowledge will enable future work to design strategies to inhibit ArnA and thus block the resistance pathway.Support or Funding InformationNational Institutes of Health (NIH) Grant # 5SC3GM118199This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.