Characterization of the Acyl-Coenzyme A Synthetase AtuH in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a common bacterium that can cause infections in both animals and plants. The bacterium, an opportunistic pathogen with antibiotic resistance, is a large contributor to hospital infections, including those affecting patients with cystic fibrosis. P. aeruginosa is a member of the Pseudomonadacae family and can utilize a variety of organic compounds as carbon sources, including acyclic terpenes. One pathway found in P. aeruginosa is the Acyclic Terpene Utilization (Atu) pathway that is partly responsible for the metabolism of citronellol and geraniol into the downfield products acetyl-CoA and acetoactate. AtuH, annotated as a long chain acyl-CoA synthetase, is a member of the AMP-forming family of enzymes. In P. aeruginosa, AtuH is proposed to be responsible for the conversion of citronellate to cintronellyl-CoA through a two-step reaction involving the utilization of ATP to form an acyl-AMP intermediate before forming the acyl-CoA in the second step. The focus of this work is to characterize AtuH in terms of its biological function and range as well as its efficiency as an enzyme. This will include cloning, expression, and purification of the enzyme using various techniques of molecular biology followed by kinetic assays with a panel of carboxylic acid derivatives of various length, saturation and substitution. The results of this work will characterize AtuH, help prove or disprove its involvement in the Atu pathway, and expand our general knowledge of acyl-CoA synthetases and their importance in biological systems.