Tight association of N-terminal and catalytic subunits of rabbit 12/15-lipoxygenase is important for protein stability and catalytic activity

Igor Ivanov, Almerinda Di Venere, Thomas Horn, Patrick Scheerer, Eleonora Nicolai, Sabine Stehling, Constanze Richter, Ewa Skrzypczak-Jankun, Giampiero Mei, Mauro MacCarrone, Hartmut Kühn

Research output: Contribution to journalArticlepeer-review

Abstract

12/15-Lipoxygenases (12/15-LOXs) have been implicated in inflammatory and hyperproliferative diseases but the structural biology of these enzymes is not well developed. Most LOXs constitute single polypeptide chain proteins that fold into a two-domain structure. In the crystal structure the two domains are tightly associated, but small angle X-ray scattering data and dynamic fluorescence studies suggested a high degree of structural flexibility involving movement of the N-terminal domain relative to catalytic subunit. When we inspected the interdomain interface we have found a limited number of side-chain contacts which are involved in interactions of these two structural subunits. One of such contact points involves tyrosine 98 of N-terminal domain. This aromatic amino acid is invariant in vertebrate LOXs regardless of overall sequence identity. To explore in more detail the role of aromatic interactions in interdomain association we have mutated Y98 to various residues and quantified the structural and functional consequences of these alterations. We have found that loss of an aromatic moiety at position 98 impaired the catalytic activity and membrane binding capacity of the mutant enzymes. Although CD and fluorescence emission spectra of wild-type and mutant enzyme species were indistinguishable, the mutation led to enlargement of the molecular shape of the enzyme as detected by analytic gel filtration and this structural alteration was shown to be associated with a loss of protein thermal stability. The possible role of tight interdomain association for the enzyme's structural performance is discussed.

Original languageEnglish
Pages (from-to)1001-1010
Number of pages10
JournalBiochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
Volume1811
Issue number12
DOIs
Publication statusPublished - Dec 2011

Keywords

  • Eicosanoids
  • Lipid peroxidation
  • Molecular enzymology
  • Motional flexibility
  • Structural biology
  • Thermal stability

ASJC Scopus subject areas

  • Cell Biology
  • Molecular Biology

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