Organism-Adapted Specificity of the Allosteric Regulation of Pyruvate Kinase in Lactic Acid Bacteria

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.

Allosteric pyruvate kinase-based “logic gate” synergistically senses energy and sugar levels in Mycobacterium tuberculosis

“…In addition, the enzyme catalytic efficiency, which is estimated by k cat / S 0.5 value (Table 1 ), is also enhanced by the synergy of AMP and G6P. In contrast to previous computational studies in which effectors were docked at a single canonical allosteric site of bacterial PYKs 24 , our kinetic results demonstrate that AMP and G6P act in synergy to allosterically activate Mtb PYK and suggest two cooperative allosteric sites. The latter was borne out in structural studies.…”

The HMGB1 protein induces a metabolic type of tumour cell death by blocking aerobic respiration

“…It is likely that HMGB1 in excess—for example, during sepsis 1 —at least partly contributes to the failure of organs through inhibition of cellular glucose-driven respiration. Our results could also explain the observation that HMGB1 can function as an important antibiotic peptide 6 by blocking the glycolytic activity of bacteria through inhibiting the PK enzyme that is highly conserved across species 20 . Thus, HMGB1 stored in secretory granules of NK cells could be an important cytotoxic compound of the innate immune system, affecting the energy metabolism of diverse pathogens and particularly of cancer cells through inhibition of tetrameric PK M2.…”

New views on an old enzyme: allosteric regulation and evolution of archaeal pyruvate kinases

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