Time-resolved structural biology as a tool to unravel and target enzyme catalysis and regulation: the actinobacterial KGD test-case

Time-resolved studies represent one of the new frontiers in the field of structural biology, where the achievable time resolution remains, in most systems, a limiting factor. We propose here to develop, in collaboration with the Crystallography Platform of the Institut Pasteur and the FIP2 beamline, time-resolved X-ray
crystallography studies on a non-photoactivable sample, specifically actinobacterial 2-oxoglutarate decarboxylase. Previous work by M. Bellinzoni’s group has
shown the features of this essential, thiamine diphosphate dependent metabolic enzyme and shown the feasibility of time-resolved studies inside crystals, making this enzyme an ideal test case. Room-temperature, humidity-controlled data collections will be initially performed, followed by time-resolved analysis following substrate injection and diffusion to isolate short-lived states in catalysis. We expect these results to define conditions for further time-resolved, serial crystallography studies.