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

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{sup 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 A4 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.
Authors:
; ; ; ; ; ; ;  [1] ;  [2] ;  [2]
  1. (Cornell)
  2. (
Publication Date:
OSTI Identifier:
1048572
Resource Type:
Journal Article
Resource Relation:
Journal Name: FASEB J.; Journal Volume: 26; Journal Issue: (8) ; 08, 2012
Research Org:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org:
NSFAHANIH
Country of Publication:
United States
Language:
ENGLISH
Subject:
60 APPLIED LIFE SCIENCES; 59 BASIC BIOLOGICAL SCIENCES; ARACHIDONIC ACID; BIOSYNTHESIS; CRYSTAL STRUCTURE; ENZYMES; GENE MUTATIONS; HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY; INFLAMMATION; LIPIDS; MACHINERY; MEMBRANES; MUTANTS; PHOSPHORYLATION; PRODUCTION; RESIDUES; SERINE; SOLVENTS; SYNTHESIS