A mutation interfering with 5-lipoxygenase domain interaction leads to increased enzyme activity.
5-Lipoxygenase (5-LOX) catalyzes two steps in conversion of arachidonic acid to proinflammatory leukotrienes. Lipoxygenases, including human 5-LOX, consist of an N-terminal C2-like β-sandwich and a catalytic domain. We expressed the 5-LOX domains separately, these were found to interact in the yeast two-hybrid system. The 5-LOX structure suggested association between Arg(101) in the β-sandwich and Asp(166) in the catalytic domain, due to electrostatic interaction as well as hydrogen bonds. Indeed, mutagenic replacements of these residues led to loss of two-hybrid interaction. Interestingly, when Arg(101) was mutated to Asp in intact 5-LOX, enzyme activity was increased. Thus, higher initial velocity of the reaction (vinit) and increased final amount of products were monitored for 5-LOX-R101D, at several different assay conditions. In the 5-LOX crystal structure, helix α2 and adjacent loops (including Asp(166)) of the 5-LOX catalytic domain has been proposed to form a flexible lid controlling access to the active site, and lid movement would be determined by bonding of lid residues to the C2-like β-sandwich. The more efficient catalysis following disruption of the R101-D166 ionic association supports the concept of such a flexible lid in human 5-LOX.
Department of Medical Biochemistry and Biophysics, Division of Physiological Chemistry II, Karolinska Institutet, S-171 77 Stockholm, Sweden.
Marija
M
Rakonjac Ryge
Department of Medical Biochemistry and Biophysics, Division of Physiological Chemistry II, Karolinska Institutet, S-171 77 Stockholm, Sweden; Department of Neurosurgery, Institute of Neurological Sciences, Faculty of Medicine, Tottori University, Yonago 6838504, Japan.
Michiharu
M
Tanabe
CHUQ Research Center/CHUL, 2705 Blvd Laurier, Quebec, QC G1V 4G2, Canada; Faculty of Medicine, Université Laval, Quebec, QC G1V 0A6, Canada.
Patrick
P
Provost
Science for Life Laboratory, Dept of Cell and Molecular Biology, Uppsala University, Box 596, S-75124 Uppsala, Sweden.
Bengt
B
Persson
Institute for Translational Medicine and Therapeutics, University of Pennsylvania, Philadelphia, PA 19104, USA.
Xinsheng
X
Chen
Institute for Translational Medicine and Therapeutics, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Biomedical and Molecular Sciences, Queen's University, Kingston, ON K7L 3N6, Canada.
Colin D
CD
Funk
Department of Medical Biochemistry and Biophysics, Division of Physiological Chemistry II, Karolinska Institutet, S-171 77 Stockholm, Sweden.
Agnes
A
Rinaldo-Matthis
Institute of Pharmaceutical Chemistry/ZAFES, University of Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany.
Bettina
B
Hofmann
Institute of Pharmaceutical Chemistry/ZAFES, University of Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany.
Dieter
D
Steinhilber
Department of Neurosurgery, Institute of Neurological Sciences, Faculty of Medicine, Tottori University, Yonago 6838504, Japan.
Takashi
T
Watanabe
Department of Medical Biochemistry and Biophysics, Division of Physiological Chemistry II, Karolinska Institutet, S-171 77 Stockholm, Sweden.
Bengt
B
Samuelsson
Department of Medical Biochemistry and Biophysics, Division of Physiological Chemistry II, Karolinska Institutet, S-171 77 Stockholm, Sweden. Electronic address: olof.radmark@ki.se.
Olof
O
Rådmark
D000595:Amino Acid Sequence; D001094:Arachidonate 5-Lipoxygenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D004789:Enzyme Activation; D006801:Humans; D015289:Leukotrienes / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017434:Protein Structure, Tertiary
D015289:Leukotrienes; D001094:Arachidonate 5-Lipoxygenase
Arachidonic acid; Domain interaction; Eicosanoid; Leukotriene; Two-hybrid system