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

被引:127
作者
Gilbert, Nathaniel C. [1 ]
Rui, Zhe [1 ]
Neau, David B. [2 ]
Waight, Maria T. [1 ]
Bartlett, Sue G. [1 ]
Boeglin, William E. [3 ,4 ]
Brash, Alan R. [3 ,4 ]
Newcomer, Marcia E. [1 ]
机构
[1] Louisiana State Univ, Dept Biol Sci, Baton Rouge, LA 70803 USA
[2] Argonne Natl Lab, NE Collaborat Access Team, Argonne, IL 60439 USA
[3] Vanderbilt Univ, Dept Pharmacol, Nashville, TN USA
[4] Vanderbilt Univ, Vanderbilt Inst Chem Biol, Nashville, TN USA
基金
美国国家科学基金会; 美国国家卫生研究院;
关键词
leukotrienes; crystal structure; crystallography; eicosanoids; ARACHIDONIC-ACID; LEUKOTRIENE BIOSYNTHESIS; CRYSTAL-STRUCTURE; HUMAN PLATELETS; PROTEIN; ACTIVATION; MODEL; 8R-LIPOXYGENASE; LIPOXYGENASES; INFLAMMATION;
D O I
10.1096/fj.12-205286
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 Ca2+-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.-Gilbert, N. C., Rui, Z., Neau, D. B., Waight, M. T., Bartlett, S. G., Boeglin, W. E., Brash, A. R., Newcomer, M. E. Conversion of human 5-lipoxygenase to a 15-lipoxygenase by a point mutation to mimic phosphorylation at Serine-663. FASEB J. 26, 3222-3229 (2012). www.fasebj.org
引用
收藏
页码:3222 / 3229
页数:8
相关论文
共 45 条
[1]  
AHARONY D, 1986, J BIOL CHEM, V261, P1512
[2]  
[Anonymous], 2002, PYMOL MOL GRAPHICS S
[3]   Aspirin-triggered lipoxin A4 and B4 analogs block extracellular signal-regulated kinase-dependent TNF-α secretion from human T cells [J].
Ariel, A ;
Chiang, N ;
Arita, M ;
Petasis, NA ;
Serhan, CN .
JOURNAL OF IMMUNOLOGY, 2003, 170 (12) :6266-6272
[4]   Intra- and extracellular expression of rabbit reticulocyte 15-lipoxygenase in the baculovirus insect cell system [J].
Borngräber, S ;
Grabenhorst, E ;
Anton, M ;
Conradt, H ;
Kühn, H .
PROTEIN EXPRESSION AND PURIFICATION, 1998, 14 (02) :237-246
[5]   Purification and catalytic activities of the two domains of the allene oxide synthase-lipoxygenase fusion protein of the coral Plexaura homomalla [J].
Boutaud, O ;
Brash, AR .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1999, 274 (47) :33764-33770
[6]   THE 3-DIMENSIONAL STRUCTURE OF AN ARACHIDONIC-ACID 15-LIPOXYGENASE [J].
BOYINGTON, JC ;
GAFFNEY, BJ ;
AMZEL, LM .
SCIENCE, 1993, 260 (5113) :1482-1486
[7]   Conformational flexibility in mammalian 15S-lipoxygenase: Reinterpretation of the crystallographic data [J].
Choi, Jongkeun ;
Chon, Jae Kyung ;
Kim, Sangsoo ;
Shin, Whanchul .
PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS, 2008, 70 (03) :1023-1032
[8]   A comprehensive model of positional and stereo control in lipoxygenases [J].
Coffa, G ;
Schneider, C ;
Brash, AR .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 2005, 338 (01) :87-92
[9]   REQUIREMENT OF A 5-LIPOXYGENASE-ACTIVATING PROTEIN FOR LEUKOTRIENE SYNTHESIS [J].
DIXON, RAF ;
DIEHL, RE ;
OPAS, E ;
RANDS, E ;
VICKERS, PJ ;
EVANS, JF ;
GILLARD, JW ;
MILLER, DK .
NATURE, 1990, 343 (6255) :282-284
[10]  
EDENIUS C, 1991, ADV PROSTAG THROMB L, V21, P97