A role of Gln596 in fine-tuning mammalian ALOX15 specificity, protein stability and allosteric properties

Alejandro Cruz, Almerinda Di Venere, Giampiero Mei, Alexander Zhuravlev, Alexey Golovanov, Sabine Stehling, Dagmar Heydeck, José M. Lluch, Àngels González-Lafont, Hartmut Kuhn, Igor Ivanov

Research output: Contribution to journalArticle

Abstract

His596 of human ALOX12 has been suggested to interact with the COO-group of arachidonic acid during ALOX catalysis. In mammalian ALOX15 orthologs Gln596 occupies this position and this amino acid exchange might contribute to the functional differences between the two ALOX-isoforms. To explore the role of Gln596 for ALOX15 functionality we mutated this amino acid to different residues in rabbit and human ALOX15 and investigated the impact of these mutations on structural, catalytic and allosteric enzyme properties. To shed light on the molecular basis of the observed functional alterations we performed in silico substrate docking studies and molecular dynamics simulations and also explored the impact of Gln596 exchange on the protein structure. The combined theoretical and experimental data suggest that Gln596 may not directly interact with the COO-group of arachidonic acid. In contrast, mutations at Gln596 destabilize the secondary and tertiary structure of ALOX15 orthologs, which may be related to a disturbance of the electrostatic interaction network with other amino acids in the immediate surrounding. Moreover, our MD-simulations suggest that the geometry of the dimer interface depends on the structure of substrate bound inside the substrate-binding pocket and that Gln596Ala exchange impairs the allosteric properties of the enzyme. Taken together, these data indicate the structural and functional importance of Gln596 for ALOX15 catalysis.

Original languageEnglish
Article number158680
JournalBiochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
Volume1865
Issue number7
DOIs
StatePublished - Jul 2020
Externally publishedYes

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Keywords

  • Allosterism
  • Fluorescence studies
  • Lipoxygenases
  • Molecular dynamics
  • Protein–protein interactions
  • Secondary structure

Cite this

Cruz, A., Di Venere, A., Mei, G., Zhuravlev, A., Golovanov, A., Stehling, S., Heydeck, D., Lluch, J. M., González-Lafont, À., Kuhn, H., & Ivanov, I. (2020). A role of Gln596 in fine-tuning mammalian ALOX15 specificity, protein stability and allosteric properties. Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, 1865(7), [158680]. https://doi.org/10.1016/j.bbalip.2020.158680