Mutagenesis of some conserved residues in human 5-lipoxygenase: effects on enzyme activity.
Recombinant human 5-lipoxygenase (arachidonate:oxygen 5-oxidoreductase, EC 1.13.11.34) was expressed in Escherichia coli. In incubations of E. coli supernatants with arachidonic acid, 5-hydroxy-7,9,11,14-eicosatetraenoic acid and leukotriene A4 were formed, while incubation with 8,11,14-eicosatrienoic acid gave 8-hydroxy-9,11,14-eicosatrienoic acid. Six conserved histidine residues in 5-lipoxygenase were subjected to site-directed mutagenesis. Exchanges of His-367, -372, or -551 gave mutants for which no enzyme activities were detectable. On the other hand, exchanges of His-362, -390, or -399 gave mutants that were enzymatically active, but less so than the nonmutated control. For two of these (exchanges of His-390 or -399), the activities of the mutants were dependent on the expression temperature. Thus, the histidines in the first group (His-367, -372, -551) were crucial for 5-lipoxygenase activity, possibly because of a function of these residues as metal ligands. Mutagenesis aimed at two other conserved elements in 5-lipoxygenase, Gln-558 and the C terminus, gave mutated proteins with only a small residual activity (substitution of Gln-558), or with no detectable activity (deletion of six C-terminal amino acids), indicating that these regions are important for the function of 5-lipoxygenase.
Zhang
Y Y
YY
Department of Physiological Chemistry, Karolinska Institutet, Stockholm, Sweden.
Rådmark
O
O
Samuelsson
B
B
D001094:Arachidonate 5-Lipoxygenase / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D001091:Arachidonate Lipoxygenases / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D004252:DNA Mutational Analysis; D015870:Gene Expression; D006639:Histidine / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D011994:Recombinant Proteins; D006639:Histidine; D001091:Arachidonate Lipoxygenases; D001094:Arachidonate 5-Lipoxygenase; C054192:arachidonate 8-lipoxygenase