Complex and reactive metallocofactors are often responsible for challenging chemistry that is critical to cellular metabolism. It is therefore not surprising that the transport of these cofactors to their target enzymes often involves multiple proteins and complex delivery pathways. Mutations in any of these components may result in a breakdown of the pathway causing deficits in the overall activity of the target enzyme and leading to a disease state, as in the case with the vitamin B12 (cobalamin, Cbl) delivery pathway. In this proposal, we will study enzymes from humans, Methylobacterium extorquens and Cupriavidus metallidurans in order to determine the molecular mechanisms of Cbl transport and installation. Our approach involves the use of a variety of biophysical methods that will allow us to visualize the interactions between a G-protein chaperone and its target Cbl- dependent mutase in the final step of the Cbl delivery pathway. This work will inform on the structural basis of chaperone-mediated delivery and repair of Cbl. By combining X-ray crystallography, electron microscopy and electron paramagnetic resonance spectroscopy, we will explore the molecular mechanism of action of the G- protein chaperones involved in the installation and repair of adenosylcobalamin in their target mutases.
Understanding the molecular mechanisms of the G-protein chaperone that modulates the final step in the pathway of transport, delivery and repair of vitamin B12 (cobalamin) into the target mutase is important in understanding how complex, and often reactive, metallocofactors are protected as they are installed into their target enzymes. This proposal uses X-ray crystallography, electron microscopy and electron paramagnetic resonance spectroscopy to explore the conformational changes of the G-protein chaperone that modulates the final step of adenosylcobalamin delivery to its target mutase, providing insight into the molecular basis for its observed function and associated human disease states.