title: Mutation analysis of the human 5-lipoxygenase C-terminus: support for a stabilizing C-terminal loop.
abstract: Lipoxygenases contain prosthetic iron, in human 5-lipoxygenase (5LO) the C-terminal isoleucine carboxylate constitutes one of five identified ligands. ATP is one of several factors determining 5LO activity. We compared properties of a series of 5LO C-terminal deletion mutants (one to six amino acid residues deleted). All mutants were enzymatically inactive (expected due to loss of iron), but expression yield (in E. coli) and affinity to ATP-agarose was markedly different. Deletion of up to four C-terminal residues was compatible with good expression and retained affinity to the ATP-column, as for wild-type 5LO. However when also the fifth residue was deleted (Asn-669) expression yield decreased and the affinity to ATP was markedly diminished. This was interpreted as a result of deranged structure and stability, due to loss of a hydrogen bond between Asn-669 and His-399. Mutagenesis of these residues supported this conclusion. In the structure of soybean lipoxygenase-1, a C-terminal loop was pointed out as important for correct orientation of the C-terminus. Accordingly, a hydrogen bond appears to stabilize such a C-terminal loop also in 5LO.
authors.affiliation: Department of Medical Biochemistry and Biophysics, Division of Physiological Chemistry II, Karolinska Institutet, S-171 77 Stockholm, Sweden.
authors.forename: Hisayo
authors.initials: H
authors.lastname: Okamoto
authors.forename: Tove
authors.initials: T
authors.lastname: Hammarberg
authors.forename: Ying-Yi
authors.initials: YY
authors.lastname: Zhang
authors.forename: Bengt
authors.initials: B
authors.lastname: Persson
authors.forename: Takashi
authors.initials: T
authors.lastname: Watanabe
authors.forename: Bengt
authors.initials: B
authors.lastname: Samuelsson
authors.forename: Olof
authors.initials: O
authors.lastname: Rådmark
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D001094:Arachidonate 5-Lipoxygenase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004252:DNA Mutational Analysis; D006801:Humans; D006860:Hydrogen Bonding; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017384:Sequence Deletion; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics
chemical_list: D014364:Tryptophan; D000255:Adenosine Triphosphate; D001094:Arachidonate 5-Lipoxygenase