Characterization of metal binding of bifunctional kinase/phosphatase AceK and implication in activity modulation

Sci Rep. 2019 Jun 24;9(1):9198. doi: 10.1038/s41598-019-45704-z.

Abstract

A unique bifunctional enzyme, isocitrate dehydrogenase kinase/phosphatase (AceK) regulates isocitrate dehydrogenase (IDH) by phosphorylation and dephosphorylation in response to nutrient availability. Herein we report the crystal structure of AceK in complex with ADP and Mn2+ ions. Although the overall structure is similar to the previously reported structures which contain only one Mg2+ ion, surprisingly, two Mn2+ ions are found in the catalytic center of the AceK-Mn2+ structure. Our enzymatic assays demonstrate that AceK-Mn2+ showed higher phosphatase activity than AceK-Mg2+, whereas the kinase activity was relatively unaffected. We created mutants of AceK for all metal-coordinating residues. The phosphatase activities of these mutants were significantly impaired, suggesting the pivotal role of the binuclear (M1-M2) core in AceK phosphatase catalysis. Moreover, we have studied the interactions of Mn2+ and Mg2+ with wild-type and mutant AceK and found that the number of metal ions bound to AceK is in full agreement with the crystal structures. Combined with the enzymatic results, we demonstrate that AceK exhibits phosphatase activity in the presence of two, but not one, Mn2+ ions, similar to PPM phosphatases. Taken together, we suggest that metal ions help AceK to balance and fine tune its kinase and phosphatase activities.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Diphosphate / metabolism*
  • Catalytic Domain
  • Escherichia coli / metabolism
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism*
  • Isocitrate Dehydrogenase / genetics
  • Isocitrate Dehydrogenase / metabolism*
  • Kinetics
  • Magnesium / metabolism*
  • Manganese / metabolism*
  • Models, Molecular
  • Mutation
  • Protein Binding

Substances

  • Escherichia coli Proteins
  • Manganese
  • Adenosine Diphosphate
  • AceK protein, E coli
  • Isocitrate Dehydrogenase
  • Magnesium

Grant support