Uncovering Condensation Domain Substrate Specificity and Structural Mechanism Using Wide-Angle X-Ray Scattering and Protein Crystallography
In recent years the human gut microbiome has gained the attention of many researchers due to its role in overall health and the accumulation of metagenomic data. The human gut microbiome has been linked to cardiovascular disease, irritable bowel disease, and several types of cancer. Gut bacteria have been shown to produce molecules that affect these GPCRs associated with these diseases. Understanding the proteins involved in their synthesis could lead to therapeutics that aim at regulating their production and the dysfunctionality they may be causing. The bacterium Corprococcus eutactus which is found in the human gut produces the fatty acid amide oleoyl aminovaleric acid which effects GPCRs. It is synthesized in the bacterium via non-ribosomal peptide synthetases (NRPS). NRPS are multi-domain modular enzymes that catalyze the synthesis of small molecules using amino acids or non-proteinogenic amino acid substrates. This occurs in an assembly line process until a functional molecule is formed and completely constructed. The 3 core domains that all NRPS contain include condensation, adenylation, and acyl carrier protein. Unlike canonical NRPS which have multidomain architectures, each domain from C. eucactus exists as its own independent protein. Due to the minimalistic nature of this NRPS, the condensation domain from this pathway (OaaC) is not only responsible for the condensation reaction but also for substrate specificity. Condensation domain substrate specificity has remained elusive thus my research focuses on uncovering sequence motifs responsible for this in OaaC as well as understanding the overall structural mechanism. I have chosen to approach this problem from two angles; one of which involves of using solution scattering and the other protein X-ray crystallography. With this approach I am able to uniquely study OaaC in its crystalline form as well its solution state allowing me to obtain high-resolution information about substrate binding and any conformational changes that are associated with this. All together these studies will elucidate substrate specificity along with the structural mechanism for OaaC which are extendable to other NRPS gene clusters that are organized similarly.