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Magnesium is an important divalent cation for the regulation of catalytic activity. Recently, we have described that the Mg 2+ binding through the PAS domain inhibits the phosphoglycerate kinase (PGK) activity in PAS domain-containing PGK from Leishmania major (LmPAS-PGK) at neutral pH 7.5, but PGK activity is derepressed at acidic pH 5.5. The acidic residue within the PAS domain of LmPAS-PGK is expected to bind the cofactor Mg 2+ ion at neutral pH, but which specific acidic residue(s) is/are responsible for the Mg 2+ binding is still unknown. To identify the residues, we exploited mutational studies of all acidic (twelve Asp/Glu) residues in the PAS domain for plausible Mg 2+ binding. Mg 2+ ion-dependent repression at pH 7.5 is withdrawn by substitution of Asp-4 with Ala, whereas other acidic residue mutants (D16A, D22A, D24A, D29A, D43A, D44A, D60A, D63A, D77A, D87A, and E107A) showed similar features compared to the wild-type protein. Fluorescence spectroscopic studies and isothermal titration calorimetry analysis showed that the Asp-4 is crucial for Mg 2+ binding in the absence of both PGK's substrates. These results suggest that Asp-4 residue in the regulatory (PAS) domain of wild type enzymes is required for Mg 2+ dependent repressed state of the catalytic PGK domain at neutral pH., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Recently, we described the PAS domain-containing phosphoglycerate kinase (PGK) from Leishmania major (LmPAS-PGK) that shows acidic pH (5.5)-dependent optimum catalytic activity. The PAS domain of LmPAS-PGK is expected to regulate PGK activity during catalysis, but the mechanism of regulation by PAS domain at the molecular level is uncharacterized. In this work, we have utilized the full-length, PAS domain-deleted, and mutant enzymes to measure the enzymatic activity in the presence of divalent cation at various pH values. Catalytic activity measurement indicates that Mg 2+ binding through PAS domain inhibits the PGK activity at pH 7.5, and this inhibition is withdrawn at pH 5.5. To identify the Mg 2+ binding residues of the PAS domain, we exploited a systematic mutational analysis of all (four) His residues in the PAS domain for potential divalent cation binding. Replacement of His-57 with alanine resulted in depression in the presence of Mg 2+ at pH 7.5, but H71A, H89A, and H111A showed similar characteristics with respect to the wild-type protein. Fluorescence and isothermal titration calorimetry studies revealed that H57 is responsible for Mg 2+ binding in the absence of substrates. Thus, the protonated form of His57 at acidic pH 5.5 destabilizes the Mg 2+ binding in the PAS domain, which is an essential requirement in the wild-type LmPAS-PGK for a conformational alteration in the sensor domain that, sequentially, activates the PGK domain, resulting in the synthesis of higher amounts of ATP., (© 2020 Federation of European Biochemical Societies.)

Per-Arnt-Sim (PAS) domains are structurally conserved and present in numerous proteins throughout all branches of the phylogenetic tree. Although PAS domain-containing proteins are major players for the adaptation to environmental stimuli in both prokaryotic and eukaryotic organisms, these types of proteins are still uncharacterized in the trypanosomatid parasites, Trypanosome and Leishmania In addition, PAS-containing phosphoglycerate kinase (PGK) protein is uncharacterized in the literature. Here, we report a PAS domain-containing PGK (LmPAS-PGK) in the unicellular pathogen Leishmania The modeled structure of N-terminal of this protein exhibits four antiparallel β sheets centrally flanked by α helices, which is similar to the characteristic signature of PAS domain. Activity measurements suggest that acidic pH can directly stimulate PGK activity. Localization studies demonstrate that the protein is highly enriched in the glycosome and its presence can also be seen in the lysosome. Gene knockout, overexpression and complement studies suggest that LmPAS-PGK plays a fundamental role in cell survival through autophagy. Furthermore, the knockout cells display a marked decrease in virulence when host macrophage and BALB/c mice were infected with them. Our work begins to clarify how acidic pH-dependent ATP generation by PGK is likely to function in cellular adaptability of Leishmania ., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Recently, we described the PAS domain‐containing phosphoglycerate kinase (PGK) from Leishmania major (LmPAS‐PGK) that shows acidic pH (5.5)‐dependent optimum catalytic activity. The PAS domain of LmPAS‐PGK is expected to regulate PGK activity during catalysis, but the mechanism of regulation by PAS domain at the molecular level is uncharacterized. In this work, we have utilized the full‐length, PAS domain‐deleted, and mutant enzymes to measure the enzymatic activity in the presence of divalent cation at various pH values. Catalytic activity measurement indicates that Mg2+ binding through PAS domain inhibits the PGK activity at pH 7.5, and this inhibition is withdrawn at pH 5.5. To identify the Mg2+ binding residues of the PAS domain, we exploited a systematic mutational analysis of all (four) His residues in the PAS domain for potential divalent cation binding. Replacement of His‐57 with alanine resulted in depression in the presence of Mg2+ at pH 7.5, but H71A, H89A, and H111A showed similar characteristics with respect to the wild‐type protein. Fluorescence and isothermal titration calorimetry studies revealed that H57 is responsible for Mg2+ binding in the absence of substrates. Thus, the protonated form of His57 at acidic pH 5.5 destabilizes the Mg2+ binding in the PAS domain, which is an essential requirement in the wild‐type LmPAS‐PGK for a conformational alteration in the sensor domain that, sequentially, activates the PGK domain, resulting in the synthesis of higher amounts of ATP. [ABSTRACT FROM AUTHOR]