TY - JOUR
T1 - A role of Gln596 in fine-tuning mammalian ALOX15 specificity, protein stability and allosteric properties
AU - Cruz, Alejandro
AU - Di Venere, Almerinda
AU - Mei, Giampiero
AU - Zhuravlev, Alexander
AU - Golovanov, Alexey
AU - Stehling, Sabine
AU - Heydeck, Dagmar
AU - Lluch, José M.
AU - González-Lafont, Àngels
AU - Kuhn, Hartmut
AU - Ivanov, Igor
N1 - Publisher Copyright:
© 2020 Elsevier B.V.
PY - 2020/7
Y1 - 2020/7
N2 - 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.
AB - 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.
KW - Allosterism
KW - Fluorescence studies
KW - Lipoxygenases
KW - Molecular dynamics
KW - Protein–protein interactions
KW - Secondary structure
UR - http://www.scopus.com/inward/record.url?scp=85082184144&partnerID=8YFLogxK
U2 - 10.1016/j.bbalip.2020.158680
DO - 10.1016/j.bbalip.2020.158680
M3 - Artículo
C2 - 32151768
AN - SCOPUS:85082184144
SN - 1388-1981
VL - 1865
JO - Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
JF - Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
IS - 7
M1 - 158680
ER -