Mutagenesis of Sequence Determinants of Truncated Porcine ALOX15 Induces Changes in the Reaction Specificity by Altering the Catalytic Mechanism of Initial Hydrogen Abstraction

Patricia Saura, Ilya Kaganer, Dagmar Heydeck, José M. Lluch, Hartmut Kühn, Àngels González-Lafont

Research output: Contribution to journalArticleResearchpeer-review

6 Citations (Scopus)

Abstract

© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim The reaction specificity of lipoxygenases is of physiological relevance since the various oxygenation products exhibit different biological activities. Among mammalian ALOX15 orthologs there are arachidonic acid 12- and 15-lipoxygenating enzymes and recent studies suggested an evolutionary switch in that reaction specificity during late primate development. Previous reports showed that 12-lipoxygenating ALOX15 orthologs can be converted to 15-lipoxygenating enzymes by site-directed mutagenesis of some sequence determinants. Unfortunately, the molecular basis for those alterations are not well understood. Here, the arachidonic acid 12-lipoxygenating N-terminal truncation variant of pig ALOX15, for which a crystal structure is available, was used to explore the catalytic mechanism of the specificity switch induced by mutagenesis of Val418 and Val419 sequence determinants. We found that Val418Ile+Val419Met double mutant is dominantly 15-lipoxygenating. Docking and MD simulations, and quantum mechanics/molecular mechanics calculations indicated that the wildtype energy barrier for arachidonic acid 15-lipoxygenation is 3.4 kcal mol−1 higher than for 12-lipoxygenation. In contrast, for the Val418Ile+Val419Met double mutant the energy barrier for 12-lipoxygenation is 6.0 kcal mol−1 higher than for 15-lipoxygenation. Our data suggest that enzyme–substrate complex geometries determine the value of these energy barriers and, as a consequence, the reaction specificity of ALOX15 orthologs.
Original languageEnglish
Pages (from-to)962-973
JournalChemistry - A European Journal
Volume24
Issue number4
DOIs
Publication statusPublished - 19 Jan 2018

Keywords

  • biological chemistry
  • enzymes
  • evolution
  • mammals
  • oxygenation

Fingerprint Dive into the research topics of 'Mutagenesis of Sequence Determinants of Truncated Porcine ALOX15 Induces Changes in the Reaction Specificity by Altering the Catalytic Mechanism of Initial Hydrogen Abstraction'. Together they form a unique fingerprint.

Cite this