Uesugi Yoshiko, Arima Jiro, Iwabuchi Masaki, Hatanaka Tadashi
Research Institute for Biological Sciences (RIBS), Okayama, Japan.
FEBS J. 2007 May;274(10):2672-81. doi: 10.1111/j.1742-4658.2007.05802.x. Epub 2007 Apr 25.
Recently, we identified Ala426 and Lys438 of phospholipase D from Streptomyces septatus TH-2 (TH-2PLD) as important residues for activity, stability and selectivity in transphosphatidylation. These residues are located in a C-terminal flexible loop separate from two catalytic HxKxxxxD motifs. To study the role of these residues in substrate recognition, we evaluated the affinities of inactive mutants, in which these residues were substituted with Phe and His, toward several phospholipids by SPR analysis. By substituting Ala426 and Lys438 with Phe and His, respectively, the inactive mutant showed a much stronger interaction with phosphatidylcholine and a weaker interaction with phosphatidylglycerol than the inactive TH-2PLD mutant. We demonstrated that Ala426 and Lys438 of TH-2PLD play a role in sensing the head group of phospholipids.
最近，我们确定了来自龟裂链霉菌TH-2（TH-2PLD）的磷脂酶D的Ala426和Lys438是反磷脂酰化活性、稳定性和选择性的重要残基。这些残基位于与两个催化性HxKxxxxD基序分开的C端柔性环中。为了研究这些残基在底物识别中的作用，我们通过表面等离子体共振（SPR）分析评估了这些残基被苯丙氨酸和组氨酸取代的无活性突变体对几种磷脂的亲和力。通过分别用苯丙氨酸和组氨酸取代Ala426和Lys438，与无活性的TH-2PLD突变体相比，该无活性突变体与磷脂酰胆碱的相互作用更强，与磷脂酰甘油的相互作用更弱。我们证明了TH-2PLD的Ala426和Lys438在感知磷脂头部基团中发挥作用。
