Exploring Phosphofructokinase Evolution

<p><strong>Phosphofructokinase (PFK) is a key enzyme in the central metabolic pathway of glycolysis. In this thesis, I investigate patterns of PFK evolution by horizontal gene transfer, and expand current knowledge of biophysical, biochemical, and structural properties of PFKs. There are three categories of PFK depending on which phosphate donor is used: ATP, ADP, or inorganic pyrophosphate (PPi). These PFKs also differ in their oligomeric states and allosteric properties. Typically, eukaryotes use ATP-dependent PFKs while archaea have ADP-dependent PFKs. Despite the complexities in properties across PFKs, these enzymes are known to be horizontally transferred across the tree of life. To shed light on how this might be possible, I constructed an Escherichia coli strain lacking both its pfk genes and individually introduced three foreign pfk genes from across the tree of life (under control of the promoter for the native phosphofructokinase A). The pfk genes were from Methanosarcina acetivorans, Borrelia burgdorferi, and Candidatus Prometheoarchaeum syntrophicum, and two versions of each gene were introduced into separate strains. One was the native copy of the gene, the other a version with codons optimised for expression in E. coli. Each encoded enzyme also differed in its phosphate donor specificity. As expected, horizontal transfer resulted in a fitness defect, which was ameliorated by serial passaging with selection on glucose as the sole carbon source. In three separate lines of each strain, E. coli evolved back to near wild type fitness in less than 100 generations. Genome sequencing revealed mutations in genes encoding proteins in the citric acid cycle, transcriptional regulators of glucose and glycerol metabolism, and DNA repair. Ultimately, the foreign PFKs were most likely bypassed by increasing flux through alternative metabolic pathways such as the pentose phosphate and/or Entner-Doudoroff pathways. These results suggest that phosphate donor specificity imposes a substantial barrier for horizontal transfer of pfk genes across the tree of life.</strong></p><p>One of the foreign pfk genes investigated in horizontal transfer experiments, Ca. P. syntrophicum, was from the newly discovered Asgard archaea. This organism is currently the closest cultured prokaryotic relative to eukaryotes. Its PFK enzyme was annotated as an ATP or PPi-dependent PFK, which was surprising because most archaeal PFKs are ADP-dependent. It was therefore hypothesised that biochemical characterisation of the Ca. P. syntrophicum PFK (PsyPFK) might shed new light on the evolution of phosphofructokinases, including the evolution of phosphate donor specificity. Activity assays and isothermal titration calorimetry determined that PsyPFK uses PPi as its substrate, but not ATP or ADP. Subsequent kinetics assays found that PsyPFK is able to phosphorylate its substrate, fructose 6-phopshate, as well as a metabolite from the pentose phosphate pathway (sedoheptulose 7-phosphate), with similar catalytic efficiency. In the opposite direction, PsyPFK was found to dephosphorylate fructose 1,6-bisphosphate with higher catalytic efficiency than both kinase reactions. Therefore, PsyPFK is similar to bacterial PPi-dependent PFKs because it is a multi-functional enzyme that performs several roles across glycolysis, gluconeogenesis, and the pentose phosphate pathway. Also, the oligomeric state of PsyPFK was a tetramer, similar to ATP-dependent PFKs but unlike other PPi-dependent PFKs (typically dimers). X-ray crystallography was then used to determine the structure of PsyPFK. Three structures were refined; apo PsyPFK, PsyPFK with fructose 6-phosphate bound, and PsyPFK with phosphoenolpyruvate, Mg2+, and Pi bound. Each of these provided insight into both the phosphate donor specificity and sugar specificity of PsyPFK. Interestingly, closest structural homologues were ATP-dependent PFKs. The phylogenetic position of Ca. P. syntrophicum and the properties of PsyPFK suggest that the ATP-dependent eukaryotic PFKs may have evolved from a PPi-dependent, PsyPFK-like ancestor, and a plausible model is proposed.</p>