Journal of Biological Chemistry
Volume 294, Issue 42, 18 October 2019, Pages 15505-15516
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Article
Evolutionary plasticity in the allosteric regulator-binding site of pyruvate kinase isoform PykA from Pseudomonas aeruginosaStructure and regulation of PykA

https://doi.org/10.1074/jbc.RA119.009156Get rights and content
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Unlike many other well-characterized bacteria, the opportunistic human pathogen Pseudomonas aeruginosa relies exclusively on the Entner-Doudoroff pathway (EDP) for glycolysis. Pyruvate kinase (PK) is the main “pacemaker” of the EDP, and its activity is also relevant for P. aeruginosa virulence. Two distinct isozymes of bacterial PK have been recognized, PykA and PykF. Here, using growth and expression analyses of relevant PK mutants, we show that PykA is the dominant isoform in P. aeruginosa. Enzyme kinetics assays revealed that PykA displays potent K-type allosteric activation by glucose 6-phosphate and by intermediates from the pentose phosphate pathway. Unexpectedly, the X-ray structure of PykA at 2.4 Å resolution revealed that glucose 6-phosphate binds in a pocket that is distinct from the binding site reported for this metabolite in the PK from Mycobacterium tuberculosis (the only other available bacterial PK structure containing bound glucose 6-phosphate). We propose a mechanism by which glucose 6-phosphate binding at the allosteric site communicates with the PykA active site. Taken together, our findings indicate remarkable evolutionary plasticity in the mechanism(s) by which PK senses and responds to allosteric signals.

glycolysis
pentose phosphate pathway (PPP)
bacterial metabolism
pyruvate kinase
X-ray crystallography
Pseudomonas
allostery
Entner-Doudoroff Pathway
Pseudomonas aeruginosa
PykA

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This work was supported by a Ph.D. Studentship from the Yousef Jameel Foundation (to Y. A.), a BBSRC Studentship (to J. G.), and BBSRC Grant BB/M019411/1 . The authors declare that they have no conflicts of interest with the contents of this article.

This article contains Figs. S1–S13 and Table S1.

The atomic coordinates and structure factors (code 6QXL) have been deposited in the Protein Data Bank (http://wwpdb.org/).