Two isoforms of pyruvate kinase enzymes in Pseudomonas aeruginosa with distinct functional and structural properties

In most organisms, phosphofructokinase (PFK) and pyruvate kinase (PK) are the key glycolytic regulatory enzymes. However, the opportunistic human pathogen, Pseudomonas aeruginosa, relies entirely on the Entner-Doudoroff pathway for glycolysis, and consequently, does not encode a PFK homologue. It does encode two PK isozymes though, denoted PykA and PykF. This arrangement is uncommon in bacteria, although when it does arise, PykF is usually the dominant isozyme. In this project, I investigated the genetic, functional and structural characteristics of PykA and PykF in P. aeruginosa. The P. aeruginosa PykA and PykF enzymes are phylogenetically distinct, and display a number of unusual properties compared with the isozymes previously characterized from other species. I found that a pykA mutant (but not a pykF mutant) of P. aeruginosa showed decreased growth on glucose and glycerol, suggesting that PykA is the dominant enzyme in this pathogen. However, a mutant defective in both pykA and pykF could be complemented (i.e., made to grow normally on glucose or glycerol) by expression of either enzyme in trans, indicating that both enzymes have the potential to be active. Consistent with the notion that PykA is the dominant enzyme in P. aeruginosa, I also found that PykA (but not PykF) was highly expressed under all conditions tested. Biochemical characterization revealed that purified PykA and PykF share similar catalytic activity, but were differentially regulated by a number of metabolites, most notably by intermediates from the anabolic pentose phosphate pathway. This suggests that P. aeruginosa coordinates glycolysis with the availability of key gluconeogenic precursors, which seems to be a common emerging theme for this pathogen. Given that PykA appears to play an important physiological role, it also represents an excellent target for the development of new antimicrobial agents. With this in mind, I found that a natural product, shikonin, inhibits PykA and prevents growth on glucose. I also solved the x-ray crystal structures of P. aeruginosa PykA and PykF. The PykA structure revealed a proven regulator, glucose-6-phosphate (G6P), bound to an allosteric site and a substrate analogue, malonate, bound in the active site. Only one structure has previously been solved for a microbial PK containing a bound regulator – that of the PK from Mycobacterium tuberculosis (Mtb). Interestingly, the G6P binding site in P. aeruginosa PykA was clearly distinct from the G6P binding site in Mtb PK. Based on my results, I propose a mechanism by which the conformational change might be transmitted from the allosteric G6P site to the active site of PykA. By contrast, the P. aeruginosa PykF structure was solved in the apo-state, with no bound ligands. However, this too proved to be distinct from the structure proposed for PykF from Escherichia coli.Yousef Jameel Scholarship (Cambridge Trust