Site-directed mutagenesis studies on a putative fifth iron ligand of mouse 8S-lipoxygenase: retention of catalytic activity on mutation of serine-558 to asparagine, histidine, or alanine.
Site-directed mutagenesis studies on a putative fifth iron ligand of mouse 8S-lipoxygenase: retention of catalytic activity on mutation of serine-558 to asparagine, histidine, or alanine.
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对小鼠 8S-脂氧合酶假定的第五个铁配体的定点诱变研究：保留丝氨酸 558 突变为天冬酰胺、组氨酸或丙氨酸的催化活性。
DOI：
10.1006/abbi.2000.2175
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发表时间：
2001
影响因子：
3.9
通讯作者：
A. Brash
中科院分区：
文献类型：
--
作者：
M. Jisaka;W. Boeglin;R. Kim;A. Brash
报道的植物和动物脂氧合酶（LOX）的晶体结构表明，催化结构域中的非血红素铁由三个组氨酸（C-末端异亮氨酸）连接，并且在某些结构中还由第五个铁配体（天冬酰胺或组氨酸残基）连接。小鼠8-LOX及其同源物（例如，人15-LOX-2）的独特之处在于在该第五位置上用丝氨酸代替通常的Asn或His。为了研究该残基在小鼠8-LOX结构-功能中的重要性，使用组氨酸定向诱变将丝氨酸-558替换为天冬酰胺、组氨酸或丙氨酸。在用编码T7 RNA聚合酶的牛痘病毒感染的HeLa细胞中表达野生型小鼠8-LOX和突变体cDNA，并通过与[14 C]花生四烯酸或[14 C]亚油酸孵育，随后通过HPLC分析产物来评估它们的相对脂氧合酶活性。Ser 558 Asn和Ser 558 His突变体具有与野生型8-LOX相当或更高的活性。它们还表现出一些15-LOX活性，这表明小的结构扰动（在这种情况下是小鼠8-LOX及其15-LOX-2同源物中相同的残基）可以互换这些密切相关的酶的位置特异性。值得注意的是，Ser 558 Ala突变体表现出显著的8-LOX活性，表明该位置不是酶中的必需铁配体。我们的结论是，小鼠8-LOX是催化能力只有四个氨基酸的铁配体，和野生型酶的Ser-558不发挥催化的重要作用。
The reported crystal structures of plant and animal lipoxygenases (LOX) show that the nonheme iron in the catalytic domain is ligated by three histidines, the C-terminal isoleucine, and in certain structures also by a fifth iron ligand, an asparagine or histidine residue. Mouse 8-LOX and its homologues (e.g., human 15-LOX-2) are unique in having a serine in place of the usual Asn or His in this fifth position. To investigate the importance of the residue in mouse 8-LOX structure-function, the serine-558 was replaced by asparagine, histidine, or alanine using oligonucleotide-directed mutagenesis. Wild-type mouse 8-LOX and the mutant cDNAs were expressed in HeLa cells infected with vaccinia virus encoding T7 RNA polymerase and their relative lipoxygenase activities assessed by incubation with [14C]arachidonic acid or [14C]linoleic acid followed by HPLC analysis of the products. The Ser558Asn and Ser558His mutants had equivalent or greater activity than wild-type 8-LOX. They also exhibited some 15-LOX activity, indicating that small structural perturbations (in this case to a residue identical in mouse 8-LOX and its 15-LOX-2 homologues) can interchange the positional specificity of these closely related enzymes. Remarkably, the Ser558Ala mutant exhibited significant 8-LOX activity, indicating that this position is not an essential iron ligand in the enzyme. We conclude that mouse 8-LOX is catalytically competent with only four amino acid iron ligands, and that Ser-558 of the wild-type enzyme does not play an essential role in catalysis.
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影响因子：
2.9
作者：
Ellis, HR;Daubner, SC;Fitzpatrick, PF
通讯作者：
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DOI：
10.1073/pnas.94.12.6148
发表时间：
1997-06-10
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DOI：
10.1016/s0006-291x(88)80271-7
发表时间：
1988-12
影响因子：
3.1
作者：
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DOI：
10.1073/pnas.92.22.10064
发表时间：
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影响因子：
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