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

DOI: 10.1074/jbc.RA119.009156 DOI Help

Authors: Yassmin Abdelhamid (University of Cambridge) , Paul Brear (University of Cambridge) , Jack Greenhalgh (University of Cambridge) , Xavier Chee (University of Cambridge) , Taufiq Rahman (University of Cambridge) , Martin Welch (University of Cambridge)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of Biological Chemistry

State: Published (Approved)
Published: September 2019
Diamond Proposal Number(s): 14043

Abstract: 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.

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

Subject Areas: Biology and Bio-materials

Instruments: I04-Macromolecular Crystallography