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

N-Substituted 5-(1H-Indol-2-yl)-2-methoxyanilines Are Allosteric Inhibitors of the Linoleate Oxygenase Activity of Selected Mammalian ALOX15 Orthologs: Mechanism of Action

“…Numerous contacts between the two helices α2 and α18 restrain the mobility of the two monomers within the ALOX15 dimer. Mutagenesis studies suggested that rearrangements of intramolecular contacts of helix 18 may correlate with the volume of the putative substrate binding cavity of the catalytic monomer in the ligand-bound ALOX15 dimer and, thus, may fine-tune the reaction specificity of the enzyme . This hypothesis is consistent with the recent report showing that dimerization of human ALOX12 affects the enzymatic activity of this enzyme via allosteric mechanisms …”

“…Human and rabbit ALOX15 orthologs exhibit dual positional specificity as they form both 15- and 12-hydroperoxides during AA oxygenation. , In fact, a 15- to 12-H­(p)­ETE ratio of 95.3 ± 0.2 vs 4.7 ± 0.2 and 90.2 ± 0.6 vs 9.8 ± 0.8 was previously reported for recombinant rabbit and human ALOX15, respectively . Here, we found that neither 8a nor 8b impacted the product specificity of the rabbit enzyme with AA, but the product specificity of the human enzyme was slightly altered (SI Figure S9).…”

“…For this purpose, we employed the Hodgkin lymphoma cells L1236, which constitutively express human ALOX15 at high levels . These cells oxygenate AA to a mixture of 15-HpETE and 12-HpETE in the ratio 85:15 (Figure E), which is consistent with the product specificity of purified recombinant human ALOX15 . Although somewhat higher inhibitor concentrations were required to inhibit the cellular ALOX15 activity, preferential inhibition of the LA oxygenase activity by 8b was observed ( LA/AA ratios 0.14–0.18) (Figure E).…”

“…Although human ALOX15 shares about 74% of amino acid identity with its mouse ortholog, the murine enzyme lacks Gln596 that is conserved in the ALOX15 orthologs of rabbits, humans, and other higher primates. Recently, this residue has been reported to contribute to the allosteric properties of rabbit ALOX15 . In contrast, in rodents (rat, mouse), a Leu is present at position 596 but it remains unclear whether this amino acid exchange may affect the allosteric property of the enzyme.…”

“…The ff14SB force field was used for the protein atoms. The force field parameters for AA, LA, and iron and its first coordination sphere (His361, His366, His541, His545, Ile 663, and OH – ) were taken from previous works, while the specific parameters of 8a and 8b inhibitors were developed here. The calculations to generate those specific parameters were carried out following the standard protocol in AMBER with Antechamber and Parmchk2 modules even though, as both inhibitors are far from common substrates used with AMBER, a procedure developed by MacKerell et al had to be employed to overcome some large dihedral penalties.…”