A mutation interfering with 5-lipoxygenase domain interaction leads to increased enzyme activity

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• 作者：Marija Rakonjac Ryge^a ; ¹ ; Michiharu Tanabe^a ; ^b ; ¹ ; Patrick Provost^c ; ^d ; Bengt Persson^e ; Xinsheng Chen^f ; Colin D. Funk^f ; ^g ; Agnes Rinaldo-Matthis^a ; Bettina Hofmann^h ; Dieter Steinhilber^h ; Takashi Watanabe^b ; Bengt Samuelsson^a ; Olof Rå ; dmark^a ; ^{olof.radmark@ki.se" class="auth_mail}
• 关键词：Leukotriene ; Eicosanoid ; Arachidonic acid ; Two-hybrid system ; Domain interaction
• 刊名：Archives of Biochemistry and Biophysics
• 出版年：1 March, 2014
• 年：2014
• 卷：545
• 期：Complete
• 页码：179-185
• 全文大小：854 K

文摘

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 尾-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 Arg¹⁰¹ in the 尾-sandwich and Asp¹⁶⁶ 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 Arg¹⁰¹ was mutated to Asp in intact 5-LOX, enzyme activity was increased. Thus, higher initial velocity of the reaction (v_init) and increased final amount of products were monitored for 5-LOX-R101D, at several different assay conditions. In the 5-LOX crystal structure, helix 伪2 and adjacent loops (including Asp¹⁶⁶) 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 尾-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.