期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1992
卷号：89
期号：2
页码：485-489
DOI：10.1073/pnas.89.2.485
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：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.
