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

Abstract

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 PykAPA and PykFPA. 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 PykAPA has been previously characterized in detail, so in this work, we set out to assess the biochemical and structural properties of the PykFPA isozyme. We show that pykFPA expression is induced in the presence of the diureide, allantoin. In spite of their relatively low amino acid sequence identity, PykAPA and PykFPA display broadly comparable kinetic parameters, and are allosterically regulated by a very similar set of metabolites. However, the x-ray crystal structure of PykFPA revealed significant differences compared with PykAPA. Notably, although the main allosteric regulator binding-site of PykFPA 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 PykFPA 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.

Pyruvate kinase 2 from Synechocystis sp. PCC 6803 increased substrate affinity via glucose-6-phosphate and ribose-5-phosphate for phosphoenolpyruvate consumption

“…5 b and Table 1 ). Similar to Synechococcus Pyk and other bacterial Pyks, the saturation curves of Sy Pyk2 for PEP showed sigmoidal curves (Knowles et al 2001 ; Jetten et al 1994 ; Abdelhamid et al 2019 ; 2021 ), and for ADP, hyperbolic curves (Abdelhamid et al 2019 ; 2021 ). Additionally, we measured the activity of Sy Pyk2 at 30 °C and pH 7.0; the conditions were optimal for Synechocystis PEPC (Takeya et al 2017 ) and competed with Sy Pyk2 for PEP consumption.…”

“…Pyks have been studied for their properties and primary sequences (Hunsley and Suelter 1969 ; Cottam et al 1969 ; Abdelhamid et al 2019 ; 2021 ). All Pyks require divalent cations, such as Mg 2+ or Mn 2+ , and numerous Pyks require K + for activity (Baek and Nowak 1982 ; Kachmar and Boyer 1953 ).…”

Bacteria−Based Synergistic Therapy in the Backdrop of Synthetic Biology

“…However, unattenuated live bacteria bring about safety risks and even death due to bacterial toxins when systemically administered. To be on the safe side, the virulence-related genes must be modified via transposon, gene site-directed mutation, and so forth ( 79 , 80 ) ( Table 2 ). A new type of tumstatin drug (Tum) delivery system was established by engineering Bifidobacterium .…”

Functional and structural characterization of Streptococcus pneumoniae pyruvate kinase involved in fosfomycin resistance

“…From previous studies, it is well-established that PYKs adopt a homotetrameric structure and are typically regulated allosterically by sugar phosphates, although the regulation mechanism appears to vary significantly among different species and has been the topic of ongoing research ( 12 ). For example, E. coli PykF is strongly activated by fructose 1,6-bisphosphate (FBP) while the P. aeruginosa isozyme is stimulated by glucose 6-phosphate (G6P) ( 13 , 14 ). PYK activity has been shown to affect DNA replication and cell division processes in the model Gram-positive bacterium Bacillus subtilis , highlighting the importance of regulating its activity for cell proliferation ( 15 , 16 ).…”