Site-directed mutagenesis studies on the iron-binding domain and the determinant for the substrate oxygenation site of porcine leukocyte arachidonate 12-lipoxygenase.
cDNA for arachidonate 12-lipoxygenase of porcine leukocytes was expressed in Escherichia coli. The recombinant 12-lipoxygenase was purified by immunoaffinity chromatography to near homogeneity with a specific activity of about 1.5 mumol/min per mg protein. Each of eight histidine residues, which were well-conserved among various mammalian lipoxygenases and presumed as ligands for non-heme iron, was substituted with leucine by site-directed mutagenesis. Each mutant enzyme was immunoaffinity-purified to near homogeneity. Mutations of His-361, -366 and -541 caused a total loss of enzyme activity, and the iron content was much lower (0.10, 0.06 and 0.06 g atom/mol protein) than that of the wild-type enzyme (0.53). Mutations of His-128 and -356 gave 159% and 162% specific activity of the wild-type enzyme, and the iron contents were 0.55 and 0.52 g atom/mol protein. Substitution of His-426 decreased the activity to 5%, but the iron content was 0.4 g atom/mol protein. The expression level of mutants at His-384 and -393 was too low to precisely determine the iron content. Taken together, His-361, -366 and -541 may play important roles for iron-binding in catalytically active 12-lipoxygenase. Since a high homology of amino acid sequence was known between porcine leukocyte 12-lipoxygenase and mammalian 15-lipoxygenases, we attempted to convert the 12-lipoxygenase to a 15-lipoxygenase. A double mutation of Val-418 and -419 to Ile and Met increased the ratio of 15- and 12-lipoxygenase activities from 0.1 to 5.7.
Suzuki
H
H
Department of Biochemistry, School of Medicine, Tokushima University, Japan.
Kishimoto
K
K
Yoshimoto
T
T
Yamamoto
S
S
Kanai
F
F
Ebina
Y
Y
Miyatake
A
A
Tanabe
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001092:Arachidonate 12-Lipoxygenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics; D007501:Iron / Q000032:analysis; D007962:Leukocytes / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry; D016415:Sequence Alignment; D013552:Swine
D011994:Recombinant Proteins; D007501:Iron; D001092:Arachidonate 12-Lipoxygenase