Three enzymes - One substrate Regulation of carbon flux through a "non-canonical" metabolic branchpoint

Pseudomonas aeruginosa is a common opportunistic pathogen. Recent work indicates that in many infection scenarios, P. aeruginosa exhibits an exquisite predilection for metabolizing fatty acids to yield acetyl-CoA. In most higher organisms, acetyl-CoA cannot be used for biomass production because the two carbon atoms which enter the TCA cycle are lost as CO2 . However, many bacteria are able to bypass these oxidative decarboxylation steps, allowing them to conserve carbon for gluconeogenesis. They perform this by using the “glyoxylate shunt”. Here, isocitrate is cleaved by isocitrate lyase (ICL) to yield succinate and glyoxylate (which, in a subsequent reaction, is combined with a further acetyl-CoA unit to yield the gluconeogenic precursor, malate). However, ICL has to compete with the TCA cycle enzyme, isocitrate dehydrogenase (ICD), for the available isocitrate, and it is the outcome of this “metabolic tussle” which dictates the flux of carbon through the glyoxylate shunt. In E. coli, ICD is inactivated by AceK-dependent phosphorylation, allowing flux through the glyoxylate shunt. However, P. aeruginosa is “wired up” differently because it employs not one, but two highly-expressed isocitrate dehydrogenases (ICD and IDH). For this PhD project, I focused on these three enzymes (ICD, IDH and ICL). I cloned, overexpressed and purified them at high yield to perform a thorough investigation of their kinetics, regulation and more interestingly crystal structures. I found that only one of these (the E. coli-like ICD) is regulated by AceK-mediated phosphorylation. The other, IDH, is allosterically regulated, as is the isocitrate lyase. These findings demonstrate that in P. aeruginosa the rerouting of the carbon flux through the glyoxylate shunt is delicately regulated via allostery mainly. The conditions in which the cells grow and access to either poor or rich carbon sources heavily influence the partitioning of the central metabolism. In P. aeruginosa, the TCA cycle remains more active (than in E. coli for example) even during growth on poor nutrient and this is probably an important aspect to manage oxidative stress accompanying growth. Finally, I have solved the x-ray crystal structures of ICD, IDH and ICL. These are entirely novel structures that have not been defined previously and are new entries to the Protein Data Bank. The structure solving work highlighted very interesting peculiarities to these enzymes when compared with other bacterial pathogens. This emphasizes the growing idea that Pseudomonas aeruginosa is a unique bacterium that cannot be modelled by the well-studied Escherichia coli. All this work crystallizes the knowledge to build up a picture of how flux is likely to be regulated at this “non- canonical” metabolic branchpoint and features new interesting directions for downstream applications such as drug-design.Cambridge European Scholarship Cambridge Philosophical Society Research Studentshi