School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, China.
School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China.
Int J Mol Sci. 2021 Mar 22;22(6):3219. doi: 10.3390/ijms22063219.
Phospholipases D (PLDs) play important roles in different organisms and in vitro phospholipid modifications, which attract strong interests for investigation. However, the lack of PLD structural information has seriously hampered both the understanding of their structure-function relationships and the structure-based bioengineering of this enzyme. Herein, we presented the crystal structure of a PLD from the plant-associated bacteria strain AS9 (SpPLD) at a resolution of 1.79 Å. Two classical HxKxxxxD (HKD) motifs were found in SpPLD and have shown high structural consistence with several PLDs in the same family. While comparing the structure of SpPLD with the previous resolved PLDs from the same family, several unique conformations on the C-terminus of the HKD motif were demonstrated to participate in the arrangement of the catalytic pocket of SpPLD. In SpPLD, an extented loop conformation between β9 and α9 (aa228-246) was found. Moreover, electrostatic surface potential showed that this loop region in SpPLD was positively charged while the corresponding loops in the two originated PLDs (PDB ID: 1F0I, 2ZE4/2ZE9) were neutral. The shortened loop between α10 and α11 (aa272-275) made the SpPLD unable to form the gate-like structure which existed specically in the two originated PLDs (PDB ID: 1F0I, 2ZE4/2ZE9) and functioned to stabilize the substrates. In contrast, the shortened loop conformation at this corresponding segment was more alike to several nucleases (Nuc, Zuc, mZuc, NucT) within the same family. Moreover, the loop composition between β11 and β12 was also different from the two originated PLDs (PDB ID: 1F0I, 2ZE4/2ZE9), which formed the entrance of the catalytic pocket and were closely related to substrate recognition. So far, SpPLD was the only structurally characterized PLD enzyme from . The structural information derived here not only helps for the understanding of the biological function of this enzyme in plant protection, but also helps for the understanding of the rational design of the mutant, with potential application in phospholipid modification.
磷脂酶 D（PLD）在不同的生物体和体外磷脂修饰中发挥着重要作用，这引起了人们强烈的研究兴趣。然而，缺乏 PLD 的结构信息严重阻碍了人们对其结构-功能关系的理解，以及该酶的基于结构的生物工程。在此，我们展示了一株植物相关细菌 菌株 AS9（SpPLD）PLD 的晶体结构，分辨率为 1.79 Å。在 SpPLD 中发现了两个经典的 HxKxxxxD（HKD）基序，并且与同一家族中的几个 PLD 具有高度的结构一致性。在将 SpPLD 的结构与同一家族中以前解析的 PLD 进行比较时，证明 HKD 基序的 C 末端的几个独特构象参与了 SpPLD 催化口袋的排列。在 SpPLD 中，发现β9 和α9 之间的延伸环构象（aa228-246）。此外，静电表面电势表明，SpPLD 中的该环区域带正电荷，而两个 起源的 PLD（PDB ID：1F0I、2ZE4/2ZE9）中的相应环为中性。α10 和α11 之间的缩短环（aa272-275）使 SpPLD 无法形成特定存在于两个 起源的 PLD（PDB ID：1F0I、2ZE4/2ZE9）中的类似门的结构，该结构有助于稳定底物。相比之下，该对应片段的缩短环构象更类似于同一家族中的几种核酸酶（Nuc、Zuc、mZuc、NucT）。此外，β11 和β12 之间的环组成也与两个 起源的 PLD（PDB ID：1F0I、2ZE4/2ZE9）不同，它们形成了催化口袋的入口，与底物识别密切相关。到目前为止，SpPLD 是唯一结构表征的来自 的 PLD 酶。这里获得的结构信息不仅有助于理解该酶在植物保护中的生物学功能，还有助于理解突变体的合理设计，这在磷脂修饰中具有潜在的应用。
