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首页> 外文期刊>Journal of Molecular Biology >Catalytic activity and acyl-chain selectivity of diacylglycerol kinaseεare modulated by residues in and near the lipoxygenase-like motif
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Catalytic activity and acyl-chain selectivity of diacylglycerol kinaseεare modulated by residues in and near the lipoxygenase-like motif

机译：脂氧合酶样基序内和附近的残基调节二酰基甘油激酶ε的催化活性和酰基链选择性

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摘要

Diacylglycerol kinase (DGK) ε plays an important role in the resynthesis of phosphatidylinositol by mediating the phosphorylation of diacylglycerol to phosphatidic acid. DGKεis unique among mammalian DGK isoforms in that it is the only one that shows acyl-chain selectivity, preferring diacylglycerols with an sn-2 arachidonoyl group. The region responsible for this arachidonoyl specificity is the lipoxygenase (LOX)-like motif found in the accessory domain, adjacent to DGKε's catalytic site. Many mutations within the LOX-like motif result in a loss of enzyme activity. However, the few mutants that retain significant activity exhibit some decrease in selectivity for the arachidonoyl chain. In the present work, we have explored mutations in a region adjacent to the LOX-like motif, which is also contained within the same hydrophobic segment of the protein. This adjacent region also contains a cholesterol recognition/interaction amino acid consensus motif. Being outside of the LOX-like motif, this region likely has less direct contact with the substrate, and more activity is retained with mutations. This has allowed us to probe in more detail the relationship between this region of the protein and substrate specificity. We demonstrate that this cholesterol recognition/interaction amino acid consensus domain also plays a role in acyl-chain selectivity. Despite the high degree of conservation of the amino acid sequence in this region of the protein, certain mutations result in proteins with higher activity than the wild-type protein. These mutations also result in a selective gain of acyl-chain preferences for diacylglycerols with different acyl-chain profiles. In addition to the LOX-like motif, adjacent residues also contribute to selectivity for diacylglycerols with specific acyl-chain compositions, such as those found in the phosphatidylinositol cycle.
机译：二酰基甘油激酶（DGK）ε通过介导二酰基甘油磷酸化为磷脂酸，在磷脂酰肌醇的再合成中起重要作用。 DGKε在哺乳动物DGK同工型中是独特的，因为它是唯一显示酰基链选择性的异构体，更喜欢具有sn-2花生四烯酰基的二酰基甘油。负责花生四烯酰基特异性的区域是在辅助结构域中与DGKε催化位点相邻的脂氧合酶（LOX）样基序。 LOX样基序内的许多突变导致酶活性的损失。但是，少数保留显着活性的突变体表现出对花生四烯酰基链的选择性降低。在目前的工作中，我们探索了与LOX样基序相邻的区域中的突变，该区域也包含在蛋白质的同一疏水片段中。该相邻区域还包含胆固醇识别/相互作用氨基酸共有基序。在LOX样基序之外，该区域可能与底物的直接接触较少，并且保留了更多的活性并带有突变。这使我们能够更详细地探讨蛋白质的这一区域与底物特异性之间的关系。我们证明了这种胆固醇识别/相互作用氨基酸共有结构域在酰基链选择性中也起作用。尽管在该蛋白质的该区域中氨基酸序列的高度保守，但是某些突变导致蛋白质具有比野生型蛋白质更高的活性。这些突变还导致选择性获得具有不同酰基链谱的二酰基甘油的酰基链偏好。除LOX样基序外，相邻残基也有助于具有特定酰基链组成的二酰基甘油的选择性，例如在磷脂酰肌醇循环中发现的那些。

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