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

<div><p>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 <i>Lactococcus lactis</i> 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 <i>Enterococcus faecalis</i> PYK, and to predict that the PYKs from <i>Streptococcus pyogenes</i> and <i>Lactobacillus plantarum</i> show weaker specificity for their allosteric activators, while still having fructose 1,6-bisphosphate play the main activator role <i>in vivo</i>. 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.</p></div

Molecular identification and functional characterization of the first Nα-acetyltransferase in plastids by global acetylome profiling.

International audienceProtein N(α) -terminal acetylation represents one of the most abundant protein modifications of higher eukaryotes. In humans, six N(α) -acetyltransferases (Nats) are responsible for the acetylation of approximately 80% of the cytosolic proteins. N-terminal protein acetylation has not been evidenced in organelles of metazoans, but in higher plants is a widespread modification not only in the cytosol but also in the chloroplast. In this study, we identify and characterize the first organellar-localized Nat in eukaryotes. A primary sequence-based search in Arabidopsis thaliana revealed seven putatively plastid-localized Nats of which AT2G39000 (AtNAA70) showed the highest conservation of the acetyl-CoA binding pocket. The chloroplastic localization of AtNAA70 was demonstrated by transient expression of AtNAA70:YFP in Arabidopsis mesophyll protoplasts. Homology modeling uncovered a significant conservation of tertiary structural elements between human HsNAA50 and AtNAA70. The in vivo acetylation activity of AtNAA70 was demonstrated on a number of distinct protein N(α) -termini with a newly established global acetylome profiling test after expression of AtNAA70 in E. coli. AtNAA70 predominately acetylated proteins starting with M, A, S and T, providing an explanation for most protein N-termini acetylation events found in chloroplasts. Like HsNAA50, AtNAA70 displays N(ε) -acetyltransferase activity on three internal lysine residues. All MS data have been deposited in the ProteomeXchange with identifier PXD001947 (http://proteomecentral.proteomexchange.org/dataset/PXD001947)

Section of the multiple sequence alignment of PYK showing the C-domain with the allosteric site.

<p>The boxed sequences directly contribute to the allosteric binding site. The residues in the purple boxes contribute to the phosphate binding site referred to here as 1′Pibs and those in the cyan box to 6′Pibs. The residues underlined in purple within the 1′Pibs site form a structural P-loop motif as discussed by Hirsch and colleagues <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003159#pcbi.1003159-Hirsch1" target="_blank">[30]</a>. The residues marked in orange correspond to the residues that interact with the allosteric ligand, FBP, in the <i>Saccharomyces cerevisiae</i> PYK (1A3W). The LAB PYKs show a conserved glutamate residue at the center of the allosteric site highlighted in red. In <i>Saccharomyces cerevisiae</i> PYK, it was shown experimentally that the mutation of T403 to E403 prevents allosteric activation of this PYK.</p

Computed Emodel scores of the most favourable ligand binding poses docked in the allosteric sites.

<p>Similarities in the scores of the binding poses (E_model) are observed for the PYKs of <i>Escherichia coli</i> (1PKY) and <i>Enterococcus faecalis</i> (E.fa) with FBP being the most favourable ligand for binding. The scores for the other ligands are more than 10 kcal/mol less favourable. Similarities in the scores are also found for the PYKs of <i>Streptococcus mutans</i> (S.mu), <i>Lactococcus lactis</i> (L.lac), <i>Streptococcus pyogenes</i> (S.py) and <i>Lactobacillus plantarum</i> (L.pl) which show overall weaker ligand binding with more similar scores for the different ligands. A high similarity between the scores of the <i>Lactococcus lactis</i> and <i>Streptococcus pyogenes</i> PYKs is observed.</p

Pairwise sequence identities among the PYKs from different organisms.

<p>Pairwise sequence identities among the PYKs from different organisms.</p

Allosteric binding site of PYK with the activator FBP bound.

<p>Residues V400 to V411, K446 to F470 and S481 to Q496 of the crystallographically resolved <i>Saccharomyces cerevisiae</i> PYK (PDB id: 1A3W, chain A) are shown in cartoon representation. Panel A shows possible hydrogen bonds between the FBP phosphate groups and the residues of PYK within a distance of 3 Å (all in stick representation). The structural P-loop motif (STSG) is coloured in purple. Panel B illustrates the phosphate interaction sites computed with the GRID program. The binding site of the 1′-phosphate moiety of FBP in the allosteric site is referred to as 1′Pibs and that of the 6′-phosphate moiety of FBP as 6′Pibs. The interaction energy is displayed at isocontours of −10 kcal/mol (mesh surface) and −13.5 kcal/mol (solid surface). Only the phosphate interaction sites that are located within 6 Å around FBP are shown.</p

Docking poses of the ligands with the most favourable interaction energies for two modelled PYKs.

<p>(A) G6P in the allosteric site of the <i>Streptococcus pyogenes</i> PYK. (B) FBP in the allosteric site of the <i>Enterococcus faecalis</i> PYK. The protein allosteric sites are shown in cartoon representation and the potential allosteric activators are shown in stick representation. The predicted allosteric activator FBP is bound in the allosteric site in its most favourable binding pose with a score of −81.4 kcal/mol. Hydrogen bonds between the ligands and the proteins (with a maximum length of 3.2 Å) are indicated by dashed lines.</p

Experimentally determined allosteric activators of PYKs^*.

*<p>k_0.5v values are dependent on the experimental conditions. See references for further information.</p>$<p>partial activation by R5P.</p

Quantitative comparison of the electrostatic potentials at the allosteric sites of PYKs.

<p>Identical and highly similar electrostatic potentials are indicated by red and orange colours. Light blue fields indicate no correlation. Anti-correlation of the electrostatic potentials is represented in pink, as shown in the colour key. Two clusters of different electrostatic potentials are observed. One includes the LAB, indicating almost identical electrostatic potentials in the allosteric sites of the PYKs from <i>Lactococcus lactis</i> (L.lactis_PYK) and <i>Streptococcus pyogenes</i> (S.pyogenes_PYK). Both also show high correlation to the allosteric site of the PYK from <i>Lactobacillus plantarum</i> (L.plantarum_PYK). Little correlation is observed to the other LAB. The second cluster is formed by the electrostatic potentials computed for the template (chim_temp) and the reference crystal structures (1PKY, 1A3W). As expected, almost identical electrostatic potentials are observed for the allosteric site of the chimeric template and the crystal structure 1PKY. The computed similarity indices are shown in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003159#pcbi.1003159.s006" target="_blank">Table S2</a>.</p

Electrostatic potentials at the allosteric sites of PYKs.

<p>The electrostatic potential is mapped on the molecular surface of each enzyme. Negative potentials are displayed in red, positive potentials in blue on a scale from −2 to +2 kT/e. Panel A shows a section of the crystallographically resolved <i>Saccharomyces cerevisiae</i> PYK (PDB id: 1A3W, chain A) in cartoon representation with the activator FBP bound in the allosteric site in stick representation. The electrostatic potentials displayed in panels B to D correspond to the same regions of the structure as shown in panel A. Panel B represents the electrostatic potential computed for the <i>Saccharomyces cerevisiae</i> PYK, panels C and D show the electrostatic potentials computed for the models of PYKs from <i>Streptococcus pyogenes</i> and <i>Lactobacillus plantarum</i>, respectively. The <i>Saccharomyces cerevisiae</i> PYK displays a broad region of positive potential at the allosteric binding site, whereas in the LAB PYKs, a rather negative potential is observed in parts of the allosteric binding site and in its proximity (panels C and D). These negatively charged regions may hinder electrostatic steering of allosteric activators to the allosteric site.</p