title: A conserved π-cation and an electrostatic bridge are essential for 11R-lipoxygenase catalysis and structural stability.
abstract: Lipoxygenases (LOXs) are lipid-peroxidizing enzymes that consist of a regulatory calcium- and membrane-binding PLAT (polycystin-1, lipoxygenase, α-toxin) domain and a catalytic domain. In a previous study, the crystal structure of an 11R-LOX revealed a conserved π-cation bridge connecting these two domains which could mediate the regulatory effect of the PLAT domain to the active site. Here we analyzed the role of residues Trp107 and Lys172 that constitute the π-cation bridge in 11R-LOX along with Arg106 and Asp173-a potential salt bridge, which could also contribute to the inter-domain communication. According to our kinetic assays and protein unfolding experiments conducted using differential scanning fluorimetry and circular dichroism spectroscopy, mutants with a disrupted link display diminished catalytic activity alongside reduced stability of the protein fold. The results demonstrate that both these bridges contribute to the two-domain interface, and are important for proper enzyme activation.
authors.affiliation: Department of Chemistry, Tallinn University of Technology, Akadeemia tee 15, Tallinn 12618, Estonia.
authors.forename: Priit
authors.initials: P
authors.lastname: Eek
authors.affiliation: Department of Chemistry, Tallinn University of Technology, Akadeemia tee 15, Tallinn 12618, Estonia.
authors.forename: Mari-Ann
authors.initials: MA
authors.lastname: Piht
authors.affiliation: Institute of Physics, University of Tartu, Ravila 14c, Tartu 50411, Estonia.
authors.forename: Margus
authors.initials: M
authors.lastname: Rätsep
authors.affiliation: Institute of Physics, University of Tartu, Ravila 14c, Tartu 50411, Estonia.
authors.forename: Arvi
authors.initials: A
authors.lastname: Freiberg
authors.affiliation: Department of Chemistry, Tallinn University of Technology, Akadeemia tee 15, Tallinn 12618, Estonia.
authors.forename: Ivar
authors.initials: I
authors.lastname: Järving
authors.affiliation: Department of Chemistry, Tallinn University of Technology, Akadeemia tee 15, Tallinn 12618, Estonia. Electronic address: nigulas.samel@ttu.ee.
authors.forename: Nigulas
authors.initials: N
authors.lastname: Samel
mesh_terms: D000818:Animals; D037421:Anthozoa / Q000201:enzymology*; D008084:Lipoxygenase / Q000737:chemistry*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D055672:Static Electricity
chemical_list: D008084:Lipoxygenase
keywords: Allosteric regulation; Arachidonic acid; Domain interaction; Lipoxygenase; PLAT domain; Thermal denaturation