Conversion of human 5‐lipoxygenase to a 15‐lipoxygenase by a point mutation to mimic phosphorylation at Serine‐663

Abstract

The enzyme 5-lipoxygenase (5-LOX) initiates biosynthesis of the proinflammatory leukotriene lipid mediators and, together with 15-LOX, is also required for synthesis of the anti-inflammatory lipoxins. The catalytic activity of 5-LOX is regulated through multiple mechanisms, including Ca(2+)-targeted membrane binding and phosphorylation at specific serine residues. To investigate the consequences of phosphorylation at S663, we mutated the residue to the phosphorylation mimic Asp, providing a homogenous preparation suitable for catalytic and structural studies. The S663D enzyme exhibits robust 15-LOX activity, as determined by spectrophotometric and HPLC analyses, with only traces of 5-LOX activity remaining; synthesis of the anti-inflammatory lipoxin A(4) from arachidonic acid is also detected. The crystal structure of the S663D mutant in the absence and presence of arachidonic acid (in the context of the previously reported Stable-5-LOX) reveals substantial remodeling of helices that define the active site so that the once fully encapsulated catalytic machinery is solvent accessible. Our results suggest that phosphorylation of 5-LOX at S663 could not only down-regulate leukotriene synthesis but also stimulate lipoxin production in inflammatory cells that do not express 15-LOX, thus redirecting lipid mediator biosynthesis to the production of proresolving mediators of inflammation.

Structural and dynamic insights into substrate binding and catalysis of human lipocalin prostaglandin D synthase

“…It should be noted that structures with well-defi ned binding of lipophilic ligands are relatively rare ( 49 ). Similar partial electron densities were observed for substrates in mouse urinary protein-pheromone complex ( 50 ), phospholipids in saposin B ( 53 ), and in 5-lipoxygenase-substrate complex ( 52 ).…”

Catalytic Convergence of Manganese and Iron Lipoxygenases by Replacement of a Single Amino Acid

“…In analogy, replacement of a hydrophobic amino acid at the bottom of the substrate channel of cucumber 13-LOX unmasked a positively charged residue and shifted oxygenation from C-13 to C-9 (50). This mechanism was recently highlighted by conversion of human 5-LOX to 15-LOX by replacement of Ser 663 with an Asp residue, a mimic of phosphorylation (18). The antarafacial relationship between hydrogen abstraction and oxygenation is not altered by the substrate in the opposite head-tail orientation.…”

“…Crystal structures are available for several soybean LOXs (sLOXs), coral 8R-LOX, arachidonate 15-and 5-LOX, and one prokaryotic LOX (14 -19). The binding of fatty acids to the active sites has so far with one recent exception (18) been deduced by modeling, site-directed mutagenesis, and oxidation of fatty acids with different chain lengths and numbers of double bonds (16, 20 -25).…”

Cyclin-dependent Kinase 5 Phosphorylation of Familial Prion Protein Mutants Exacerbates Conversion into Amyloid Structure

“…Nevertheless, others have observed that phosphorylation-induced conversion of several proteins is likely due to 1) the negative charge of the phosphomoiety, which can impart a repulsive charge, intramolecularly resulting in conversion of the protein (72,73); 2) the exposure of hydrophobic regions of the phosphorylated protein, leading to aggregation (74); or 3) a phosphorylation-dependent stabilization of a ␤-sheet structure (11).…”