Date of Award
Doctor of Philosophy
Biochemistry, Biophysics and Molecular Biology
Thomas A. Bobik
In contrast to conventional wisdom, bacteria are capable of forming highly organized organelles in the cytoplasm to optimize metabolic processes. Salmonella enterica can degrade the carbon source 1,2-propanediol (1,2-PD) within a proteinaceous organelle named the 1,2-PD utilization microcompartment (Pdu MCP). Prior enzymatic assays indicated that a phosphotransacylase (PTAC) was required for 1,2-PD degradation but the enzyme involved was not identified. Here we showed that the PduL enzyme is an evolutionarily distinct PTAC used for 1,2-PD degradation and that it is associated with the Pdu MCP where it plays a role in coenzyme A homeostasis. A series of genetic and growth studies showed that the house-keeping phosphotransacetylase Pta was not required for 1,2-PD metabolism in Salmonella while pduL mutants were impaired for aerobic and anaerobic growth on 1,2-PD. High-pressure liquid chromatography (HPLC) and enzymatic assays determined that PduL catalyzes the interconversion of propionyl-CoA and propionyl-PO42-. Biochemical studies indicated that PduL has kinetic properties appropriate to a role in 1,2-PD degradation. Bioinformatics analysis showed that the PduL enzyme doesn't share sequence homology with known PTAC enzymes, indicating that it represents a new enzyme class.
Recent studies suggested that the cofactors required for MCP-associated metabolism, Ado-B12 and NAD+ and HS-CoA are internally recycled within MCPs. We performed in vivo as well as in vitro studies to show that PduL is a component of the Pdu MCP and that it is used to regenerate coenzyme A inside the Pdu MCP. Western blots showed enrichment of PduL and also a PduL-green fluorescent protein (GFP) fusion in purified MCPs compared to crude cell extracts, indicating that PduL is an MCP component. Tests of genetic effects of breaking MCPs on PduL suggested that PduL is required for proper functions of intact MCPs. Further genetic studies showed that PduL in MCPs can't utilize acetyl-CoA in the cytosol to support Salmonella growth, and PduL recycling coenzyme A in the Pdu MCP is indicated. In addition, our studies also identified an N-terminal targeting sequence on PduL which mediates its encapsulation into the Pdu MCP. This short targeting sequence could also mediate the encapsulation of GFP. Together with recent publications, these studies indicate that cofactor recycling is a general mechanism used by MCPs of various types to optimize metabolic efficiency. The targeting studies advance our knowledge on spatial organization of enzymes into MCPs.
We also initiated studies on the higher order structure of the Pdu MCP by exploring the binding interactions among varied Pdu proteins. These studies identified two possible binding interactions: PduL with PduBB' and PduU with PduV. These results were consistent with an interactome network formulated using computational models. A better understanding of architecture of the Pdu MCP provide a basis for further study on its mechanisms of assembly.
Liu, Yu, "Role of the PduL enzyme in the function of primitive bacterial organelles found in Salmonella" (2014). Graduate Theses and Dissertations. 14214.