Xie Z, Ho W T, Exton J H
Howard Hughes Medical Institute and the Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0295, USA.
J Biol Chem. 1998 Dec 25;273(52):34679-82. doi: 10.1074/jbc.273.52.34679.
Rat brain phospholipase D1 (rPLD1) belongs to a superfamily defined by the highly conserved catalytic motif (H(X)K(X)4D, denoted HKD. RPLD1 contains two HKD domains, located in the N- and C-terminal regions. Deletion mutants of rPLD1 that contained only an N- or C-terminal HKD domain exhibited no catalytic activity when expressed in COS 7 cells. However, when N-terminal fragments containing one of the HKD domains were cotransfected with a C-terminal fragment containing the other HKD domain, PLD activity was restored. Furthermore, immunoprecipitation assays showed that the N- and C-terminal halves of rPLD1 were physically associated when expressed in COS 7 cells. In addition, deletion of 168 amino acids from the N terminus of rPLD1 significantly enhanced basal PLD activity while inhibiting the response to phorbol ester. Likewise, the coexpression of this truncated N-terminal half with the C-terminal half resulted in increased PLD activity. In summary, this study provides direct evidence that the enzymatic activity of rPLD1 requires the presence of the HKD domains in both the N- and C-terminal regions of the molecule. More importantly, the two halves of rPLD1 can associate, and this may be essential to bring the two HKD domains together to form an active catalytic center. These findings provide new insights into the catalytic mechanism of enzymes of the PLD superfamily.
大鼠脑磷脂酶D1（rPLD1）属于一个由高度保守的催化基序（H(X)K(X)4D，即HKD）所定义的超家族。RPLD1含有两个HKD结构域，分别位于N端和C端区域。仅含有N端或C端HKD结构域的rPLD1缺失突变体在COS 7细胞中表达时无催化活性。然而，当含有一个HKD结构域的N端片段与含有另一个HKD结构域的C端片段共转染时，PLD活性得以恢复。此外，免疫沉淀分析表明，rPLD1的N端和C端在COS 7细胞中表达时存在物理关联。另外，从rPLD1的N端缺失168个氨基酸显著增强了基础PLD活性，同时抑制了对佛波酯的反应。同样，这种截短的N端半段与C端半段共表达导致PLD活性增加。总之，本研究提供了直接证据，表明rPLD1的酶活性需要分子的N端和C端区域都存在HKD结构域。更重要的是，rPLD1的两个半段可以相互关联，这可能对于将两个HKD结构域聚集在一起形成一个活性催化中心至关重要。这些发现为PLD超家族酶的催化机制提供了新的见解。
