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

Rakonjac Ryge M, Tanabe M, Provost P, Persson B, Chen X, Funk CD, Rinaldo-Matthis A, Hofmann B, Steinhilber D, Watanabe T, Samuelsson B, Rådmark O

Arch. Biochem. Biophys. 545 (-) 179-185 [2014-03-01; online 2014-01-28]

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.

Affiliated researcher

PubMed 24480307

DOI 10.1016/j.abb.2014.01.017

Crossref 10.1016/j.abb.2014.01.017

pii: S0003-9861(14)00037-X