Evolutionary plasticity in the allosteric regulator-binding site of pyruvate kinase isoform PykA from Pseudomonas aeruginosa

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

“…Pyruvate kinase [ATP:pyruvate 2-O-phosphotransferase (EC 2.7.1.40)] catalyzes the interconversion of phosphoenolpyruvate and pyruvate in the final reaction of the "lower half " of EDP and EMP glycolysis, and is widely regarded as the main regulatory enzyme for this sequence of reactions (Rose, 1970;Kayne, 1973;Seeholzer et al, 1991;Al-Zaid Siddiquee et al, 2004;Bücker et al, 2014;Noy et al, 2016). phosphoenolpyruvate + ADP ↔ pyruvate + ATP Pseudomonas aeruginosa is among a subset of bacteria that express two distinct pyruvate kinase isoforms, denoted PykA and PykF (Waygood et al, 1975(Waygood et al, , 1976Garcia-Olalla and Garrido-Pertierra, 1987;Hofmann et al, 2013;Abdelhamid et al, 2019). Both isozymes catalyze the same reaction.…”

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“…Notably, and although the PykF sub-family of PK isozymes were originally functionally designated as being regulated by fructose 1,6-bisphosphate, this metabolite had little impact on the activity of PykF PA . Similarly, whereas the PykA family of isozymes were originally designated thus because they are regulated by AMP, this molecule had no impact on the activity of PykA PA (Abdelhamid et al, 2019) although it is a moderate activator of PykF PA . These data may suggest that assignation of a PK isozyme into the "PykA" or "PykF" sub-family, which nowadays, is largely based on sequence analyses, is a convenience that does not necessarily have any associated functional significance.…”

“…Pyruvate kinase activity was measured using a lactate dehydrogenase (LDH)-coupled assay following our previous protocol for purified PykA (Abdelhamid et al, 2019) except that unless otherwise stated, purified PykF was added to a final concentration of 0.25 µg/mL to start the reactions. Regulator screens were also carried out as previously described (Abdelhamid et al, 2019). In all experiments, regulators were added at 1 mM final concentration except for R5P, X5P, and RL5P which were used at 0.15, 0.5, and 0.5 mM, respectively.…”

“…Both isozymes catalyze the same reaction. We previously demonstrated that PykA displays potent K-type allosteric activation by glucose 6-phosphate (G6P), fructose 6-phosphate (F6P), G3P and by intermediates of the reductive pentose phosphate pathway (PPP) (Abdelhamid et al, 2019). It is important to note here that in PA, the upper half of the EDP and the upper half of the gluconeogenic pathway (encoding the aldolase, fructose 1,6-bisphosphatase, and phosphoglucoisomerase) do not operate as essentially parallel "contraflow" reactions, but instead, engage in an integrated cyclical series of reactions; the "Entner-Doudoroff-Embden-Meyerhof-Parnas" (EDEMP) cycle (Nikel et al, 2015;Kohlstedt and Wittmann, 2019).…”

“…Given that PPP intermediates strongly activate PykA, this suggests that flux through the lower half of the EDP is coordinated with the level of intermediates in the EDEMP cycle. Interestingly, the G6P-binding site in P. aeruginosa PykA (hereafter, PykA PA ) is clearly distinct from the G6P-binding site in pyruvate kinase from Mycobacterium tuberculosis, indicating remarkable plasticity in the mechanism(s) underpinning allosteric regulation in each enzyme (Zhong et al, 2017;Abdelhamid et al, 2019). On the other hand, the structure, activity and regulation of P. aeruginosa PykF (hereafter, PykF PA )…”