Understanding the Non-Ribosomal Peptide Synthetase (NRPS) system through structural and functional studies of the adenylate-forming family of enzymes
Reger, Albert S.
MetadataShow full item record
Non-Ribosomal Peptide Synthetases (NRPS) are large multi-domain enzymes that produce biologically important macromolecules. This includes peptide-derived antibiotics and siderophores that have high affinity for iron. These enzymes are made up of a number of essential and non-essential domains that function in an "assembly line" fashion to produce these small molecules. The essential domains include the condensation domain, adenylation domain, and the peptidyl-carrier domain. The non-essential domains are used to modify the growing peptide chain and for product release. This group includes the epimerization domain, methylation domain, condensation domain, and thioesterase domain. The adenylation domains of NRPSs have been a source of interest both biochemically and structurally. This domain is a family member of the adenylate-forming family of enzymes. Adenylation domains select a specific amino acid out of the cellular pool that will be integrated into the growing peptide chain. These domains like all adenylate-forming enzymes perform two unique half-reactions. The adenylate-forming half-reaction of an adenylation domain reacts an amino acid with ATP forming an adenylate intermediate. In the thioester-forming half-reaction, the adenylate is reacted with a CoA derived phosphopantheine co-factor that has been post-translational attached to the peptidyl-carrier protein. The Gulick and Dunaway-Mariano labs have used both biochemical and structural methods to further study the adenylate-forming family of enzymes. More specifically we have followed up our hypothesis that the adenylate-forming family of enzymes uses a ∼140°C-terminal domain rotation to adopt two unique conformations, where each conformation performs one of the two half-reaction. We have supported this hypothesis through mutagenesis of key residues that play a role in each half-reaction and the hinge region in bacterial Acetyl-CoA Synthetase (Acs), 4-Chlorobenzoate CoA Ligase (CBL), and FadD1 from Pseudomonas aeruginosa. We have also supported this hypothesis by solving the crystal structure of 4-Chlorobenzoate CoA Ligase in both the adenylate-forming conformation and the thioester-forming conformation with biologically relevant ligands. We have also studied the substrate binding pocket of both bacterial Acs and CBL with the goal of changing the substrate specificity of each of the enzymes. Using structural information of the individual proteins rational mutagenesis was used to expand the binding pockets to accommodate larger substrates. Specifically a V386A mutation to Acs allowed propionate to become the preferred substrate over the native substrate acetate. In CBL, an I303G mutation allowed for the improved catalytic turnover of 3,4-dichlorobenzoate. These studies show the ability to change substrate specificity is difficult, but it is not impossible.