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Mutations of Triad Determinants Changes the Substrate Alignment at the Catalytic Center of Human ALOX5
- Source :
- ACS chemical biology. 14(12)
- Publication Year :
- 2019
-
Abstract
- For the specificity of ALOX15 orthologs of different mammals the geometry of the amino acids Phe353, Ile418, Met419 and Ile593 (?triad determinants?) is important and mutagenesis of these residues altered the reaction specificity of these enzymes. Here we expressed wildtype human ALOX5 and its F359W/A424I/N425M/A603I mutant in Sf9 insect cells and characterized the catalytic differences of the two enzyme variants. We found that wildtype ALOX5 converted arachidonic acid mainly to 5(S)-HpETE. In contrast, 15(S)- and 8(S)-H(p)ETE were formed by the mutant enzyme. In addition to arachidonic acid, wildtype ALOX5 accepted EPA as substrate but C18 fatty acids were not oxygenated. The quadruple mutant also accepted linoleic acid, alpha- and gamma-linolenic acid as substrate. Structural analysis of the oxygenation products and kinetic studies with stereospecifically labeled 11(S)- and 11(R)-deutero linoleic acid suggested alternative ways of substrate orientation at the active site. In silico docking studies, molecular dynamics simulations and QM/MM calculations confirmed this hypothesis. These data indicate that ?triad determinant? mutagenesis alters the catalytic properties of ALOX5 abolishing its leukotriene synthase activity but improving its biosynthetic capacity for pro-resolving lipoxins. Fil: Ivanov, Igor. Russian Technological University; Fil: Golovanov, Alexey B.. Russian Technological University; Fil: Ferretti, Cristián Alejandro. Universidad Nacional del Litoral. Instituto de Química Aplicada del Litoral. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Química Aplicada del Litoral.; Argentina Fil: Canyelles-Niño, Miquel. Universitat Autònoma de Barcelona; España Fil: Heydeck, Dagmar. Humboldt University Berlin; Fil: Stehling, Sabine. Humboldt University Berlin; Fil: Lluch, José M.. Universitat Autònoma de Barcelona; España Fil: González Lafont, Ángels. Universitat Autònoma de Barcelona; España Fil: Kühn, Hartmut. Humboldt University Berlin
- Subjects :
- 0301 basic medicine
EICOSANOIDS
Stereochemistry
Linoleic acid
LIPOXYGENASE
01 natural sciences
Biochemistry
Molecular mechanics
Substrate Specificity
Linoleic Acid
03 medical and health sciences
chemistry.chemical_compound
Catalytic Domain
Sf9 Cells
Animals
Humans
chemistry.chemical_classification
MUTAGENESIS
Arachidonate 5-Lipoxygenase
Arachidonic Acid
biology
010405 organic chemistry
Otras Ciencias Químicas
Mutagenesis
Ciencias Químicas
Active site
Substrate (chemistry)
REACTION MECHANISM
General Medicine
0104 chemical sciences
Amino acid
Molecular Docking Simulation
Oxygen
030104 developmental biology
Enzyme
chemistry
Mutation
biology.protein
Molecular Medicine
Arachidonic acid
MOLECULAR DYNAMICS
FATTY ACIDS
CIENCIAS NATURALES Y EXACTAS
Subjects
Details
- ISSN :
- 15548937
- Volume :
- 14
- Issue :
- 12
- Database :
- OpenAIRE
- Journal :
- ACS chemical biology
- Accession number :
- edsair.doi.dedup.....544199995fe21b6b8035437353323f69