Abstract
Pyruvate kinase (PYK) is a critical allosterically regulated enzyme that links glycolysis, the primary energy metabolism, to cellular metabolism. Lactic acid bacteria rely almost exclusively on glycolysis for their energy production under anaerobic conditions, which reinforces the key role of PYK in their metabolism. These organisms are closely related, but have adapted to a huge variety of native environments. They include food-fermenting organisms, important symbionts in the human gut, and antibiotic-resistant pathogens. In contrast to the rather conserved inhibition of PYK by inorganic phosphate, the activation of PYK shows high variability in the type of activating compound between different lactic acid bacteria. System-wide comparative studies of the metabolism of lactic acid bacteria are required to understand the reasons for the diversity of these closely related microorganisms. These require knowledge of the identities of the enzyme modifiers. Here, we predict potential allosteric activators of PYKs from three lactic acid bacteria which are adapted to different native environments. We used protein structure-based molecular modeling and enzyme kinetic modeling to predict and validate potential activators of PYK. Specifically, we compared the electrostatic potential and the binding of phosphate moieties at the allosteric binding sites, and predicted potential allosteric activators by docking. We then made a kinetic model of Lactococcus lactis PYK to relate the activator predictions to the intracellular sugar-phosphate conditions in lactic acid bacteria. This strategy enabled us to predict fructose 1,6-bisphosphate as the sole activator of the Enterococcus faecalis PYK, and to predict that the PYKs from Streptococcus pyogenes and Lactobacillus plantarum show weaker specificity for their allosteric activators, while still having fructose 1,6-bisphosphate play the main activator role in vivo. These differences in the specificity of allosteric activation may reflect adaptation to different environments with different concentrations of activating compounds. The combined computational approach employed can readily be applied to other enzymes.
Highlights
Cellular metabolism comprises many different enzymatic reactions and pathways that need to work together in an orchestrated way
What makes an Lactic acid bacteria (LAB) a friend or a foe and how do they adapt to survive in such different environments? Here, we addressed this problem by focusing on the enzyme pyruvate kinase, which plays a central role in the metabolism of lactic acid bacteria
As the allosteric sites of the LAB Pyruvate kinase (PYK) are less favourable for phosphate binding than those of the Saccharomyces cerevisiae, Escherichia coli and human PYKs, we suggest that the sugar moiety of the allosteric activator plays a relatively much more critical role in binding in LAB PYKs
Summary
Cellular metabolism comprises many different enzymatic reactions and pathways that need to work together in an orchestrated way. Organisms develop complex regulatory mechanisms in order to maintain the functional balance between individual pathways. The control mechanisms need to be flexible to allow the organism to react and adapt to perturbations such as changes in the environmental conditions. Different regulatory mechanisms have developed ranging from those at the metabolic or enzyme level to those at the genetic level. A first, rapid level of response to environmental changes can be achieved by targeting the enzymatic reactions, thereby affecting enzyme activity directly [1]
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