Meruvu Sunitha, Walther Matthias, Ivanov Igor, Hammarström Sven, Fürstenberger Gerhard, Krieg Peter, Reddanna Pallu, Kuhn Hartmut
University Medicine Berlin Charité, Monbijoustrasse 2, 10117 Berlin, Germany.
J Biol Chem. 2005 Nov 4;280(44):36633-41. doi: 10.1074/jbc.M508260200. Epub 2005 Aug 29.
Mammalian lipoxygenases (LOXs) are categorized with respect to their positional specificity of arachidonic acid oxygenation. Site-directed mutagenesis identified sequence determinants for the positional specificity of these enzymes, and a critical amino acid for the stereoselectivity was recently discovered. To search for sequence determinants of murine (12R)-LOX, we carried out multiple amino acid sequence alignments and found that Phe(390), Gly(441), Ala(455), and Val(631) align with previously identified positional determinants of S-LOX isoforms. Multiple site-directed mutagenesis studies on Phe(390) and Ala(455) did not induce specific alterations in the reaction specificity, but yielded enzyme species with reduced specific activities and stereo random product patterns. Mutation of Gly(441) to Ala, which caused drastic alterations in the reaction specificity of other LOX isoforms, failed to induce major alterations in the positional specificity of mouse (12R)-LOX, but markedly modified the enantioselectivity of the enzyme. When Val(631), which aligns with the positional determinant Ile(593) of rabbit 15-LOX, was mutated to a less space-filling residue (Ala or Gly), we obtained an enzyme species with augmented catalytic activity and specifically altered reaction characteristics (major formation of chiral (11R)-hydroxyeicosatetraenoic acid methyl ester). The importance of Val(631) for the stereo control of murine (12R)-LOX was confirmed with other substrates such as methyl linoleate and 20-hydroxyeicosatetraenoic acid methyl ester. These data identify Val(631) as the major sequence determinant for the specificity of murine (12R)-LOX. Furthermore, we conclude that substrate fatty acids may adopt different catalytically productive arrangements at the active site of murine (12R)-LOX and that each of these arrangements may lead to the formation of chiral oxygenation products.
哺乳动物脂氧合酶（LOXs）根据其对花生四烯酸氧化的位置特异性进行分类。定点诱变确定了这些酶位置特异性的序列决定因素，并且最近发现了一个对立体选择性至关重要的氨基酸。为了寻找小鼠（12R）-LOX的序列决定因素，我们进行了多个氨基酸序列比对，发现苯丙氨酸（390）、甘氨酸（441）、丙氨酸（455）和缬氨酸（631）与先前确定的S-LOX亚型的位置决定因素一致。对苯丙氨酸（390）和丙氨酸（455）进行的多个定点诱变研究未在反应特异性上引起特定改变，但产生了具有降低的比活性和立体随机产物模式的酶种类。将甘氨酸（441）突变为丙氨酸，这在其他LOX亚型的反应特异性中引起了剧烈变化，但未能在小鼠（12R）-LOX的位置特异性中引起主要改变，但显著改变了该酶的对映选择性。当与兔15-LOX的位置决定因素异亮氨酸（593）对齐的缬氨酸（631）突变为空间占位较小的残基（丙氨酸或甘氨酸）时，我们获得了一种具有增强催化活性和特异性改变的反应特征（手性（11R）-羟基二十碳四烯酸甲酯的主要形成）的酶种类。用其他底物如亚油酸甲酯和20-羟基二十碳四烯酸甲酯证实了缬氨酸（631）对小鼠（12R）-LOX立体控制的重要性。这些数据确定缬氨酸（631）是小鼠（12R）-LOX特异性的主要序列决定因素。此外，我们得出结论，底物脂肪酸可能在小鼠（12R）-LOX的活性位点采取不同的催化有效排列，并且这些排列中的每一种都可能导致手性氧化产物的形成。
