Structure, Function and Regulation of a Second Pyruvate Kinase Isozyme in Pseudomonas aeruginosa

Pseudomonas aeruginosa (PA) depends on the Entner-Doudoroff pathway (EDP) for glycolysis. The main enzymatic regulator in the lower half of the EDP is pyruvate kinase. PA contains genes that encode two isoforms of pyruvate kinase, denoted PykA_PA and PykF_PA. In other well-characterized organisms containing two pyruvate kinase isoforms (such as Escherichia coli) each isozyme is differentially regulated. The structure, function and regulation of PykA_PA has been previously characterized in detail, so in this work, we set out to assess the biochemical and structural properties of the PykF_PA isozyme. We show that pykF _PA expression is induced in the presence of the diureide, allantoin. In spite of their relatively low amino acid sequence identity, PykA_PA and PykF_PA display broadly comparable kinetic parameters, and are allosterically regulated by a very similar set of metabolites. However, the x-ray crystal structure of PykF_PA revealed significant differences compared with PykA_PA. Notably, although the main allosteric regulator binding-site of PykF_PA was empty, the "ring loop" covering the site adopted a partially closed conformation. Site-directed mutation of the proline residues flanking the ring loop yielded apparent "locked on" and "locked off" allosteric activation phenotypes, depending on the residue mutated. Analysis of PykF_PA inter-protomer interactions supports a model in which the conformational transition(s) accompanying allosteric activation involve re-orientation of the A and B domains of the enzyme and subsequent closure of the active site.