The protein science research team worked with the rapeseed team to analyze plant-specific phospholipid hydrolases...

Nanhu News Network News (correspondent Fan Ruyi) Recently, Hubei Hongshan Laboratory, the National Key Laboratory of Crop Genetic Improvement, the protein science research team and the rapeseed team published an online report entitled "Insights into themechanism of phospholipid hydrolysis by plant non-specific" in Nature Communications phospholipase C"
.
This study analyzes the working mechanism of the plant-specific phospholipid hydrolase NPC4, which provides new insights
into the molecular mechanism of the eukaryotic phospholipid hydrolase family.

Nonspecific phospholipase C (NPC) is a class of plant-specific phospholipases that are induced to be expressed
under low phosphorus stress.
NPCs play an important
role in the growth and development of plants by hydrolyzing a variety of membrane phospholipids and sphingomyelins on the plasma membrane, mediating membrane lipid remodeling, recovering and utilizing inorganic phosphorus, and maintaining cellular phosphate homeostasis.
Compared with the molecular mechanism of hydrolysis substrates of other phospholipases (A1/A2/D/PI-PLC), NPTCs are the only class of phospholipases
that have not been elucidated.

Liu's main research group and Guo Liang's research group used a variety of biophysical techniques to synthesize structural analysis and dynamic characterization to reveal the molecular mechanism of plant NPC4 hydrolysis substrate (Figure 1).

The study found that the three-dimensional structure of NPC4 differs from all known phospholipases and functions in a unique structural form; The catalytic pocket is located in the phospholipase domain (PD) of NPC4, and the newly identified C-terminal domain (CTD), although it does not directly participate in the hydrolysis of the substrate, is necessary for the activity of NPC4, and stabilizes the catalytic pocket of PD through interaction with PD to form a catalytic conformation
。 Combined with biochemical analysis, the key amino acids of NPC4 hydrolyzed substrate were identified, and the enzyme-catalyzed reaction mechanism was proposed.
Through molecular docking and molecular dynamics simulation, the team further discovered that a variety of phospholipid molecules can bind to the catalytic pocket of NPC4, and then confirmed that NPC4 has low substrate specificity characteristics
at the molecular level.
This study elucidates the working mechanism of plant NPCs from the mechanism, provides new mechanisms and insights for the catalytic mechanism of phospholipase family, and provides an important reference
for the genetic improvement of efficient phosphorus utilization in plants and crops.

Figure 1.
Multi-technology combination, combined with structural basis, dynamic characterization, and biochemical analysis, elucidated the catalytic conformation of CTD-stabilized PD of NPC4, and then catalyzed the molecular mechanism
of substrate hydrolysis.

Fan Ruyi, a doctoral student, Zhao Fen, a postdoctoral fellow, and Gong Zhou, an associate researcher at the Institute of Precision Measurement Science and Technology Innovation of the Chinese Academy of Sciences, are the co-first authors of the paper, and Professor Guo Liang and Professor Liu are the co-corresponding authors
.
Professor Yin Ping of Huazhong Agricultural University, Professor Wang Xuemin of the University of Missouri-St.
Louis/Donald Danfoss Plant Science Center, and Assistant Researcher Chen Yanke of the Institute of Precision Measurement Science and Technology Innovation of the Chinese Academy of Sciences participated in the study
.
The university-scale protein platform provided strong support for this research, and the diffraction and scattering data of the samples were collected
at the Shanghai synchrotron radiation light source.
The research was supported
by the National Key Research and Development Program of China, the National Natural Science Foundation of China, and the Hubei Hongshan Laboratory Fund.

【English Abstract】

Non-specific phospholipase C (NPC) hydrolyzes major membrane phospholipids to release diacylglycerol (DAG), a potent lipid-derived messenger regulating cell functions.
Despite extensive studies on NPCs reveal their fundamental roles in plant growth and development, the mechanistic understanding of phospholipid-hydrolyzing by NPCs, remains largely unknown.
Here we report the crystal structure of Arabidopsis NPC4 at a resolution of 2.
1 Å.
NPC4 is divided into a phosphoesterase domain (PD) and a C-terminal domain (CTD), and is structurally distinct from other characterized phospholipases.
The previously uncharacterized CTD is indispensable for the full activity of NPC4.
Mechanistically, CTD contributes NPC4 activity mainly via CTDα1-PD interaction, which ultimately stabilizes the catalytic pocket in PD.
Together with a series of structure-guided biochemical studies, our work elucidates the structural basis and provides molecular mechanism of phospholipid hydrolysis by NPC4, and adds new insights into the members of phospholipase family.

Link to the paper:

Review: Liu Zhu Guo Liang