A Preliminary Study On The Formation Mechanism Of Glycerol Phosphatidylcholine During The PfGDPD Catalyzed Hydrolysis Of Lysophosphatidylcholine

Glycerophosphodiester phosphodiesterase（GDPD;EC 3.1.4.46）is a ubiquitous organism that catalyzes the hydrolysis of glycerophosphodiester to glycerol-3-phosphate and corresponding hydroxyl-containing compounds（such as choline,inositol and serine）.Based on the previous research,it was found that that GDPD（pfGDPD）derived from Pyrococcus furiosus DSM 3638 had lysophospholipase D hydrolysis activity.However,the product’s characteristics that catalyze the lysophosphatidylcholine（LPC）reaction process have not been elucidated.In this paper,³¹P-NMR and MS were used to analyze the characteristics of the products formation during the enzymatic hydrolysis of LPC,and the formation of glycerophosphatidylcholine（GPC）was observed for the first time in this process.For this unique phenomenon discovered,we try to use molecular biology and crystallography to study the formation mechanism of GPC in the enzyme-catalyzed reaction.The related research laid an essential foundation for further understanding the structure-function relationship of the enzyme and guiding its molecular modification and application.The main research contents and results are as follows:（1）³¹P-NMR analysis of the product characteristics of the hydrolysis of lysophosphatidylcholine by pfGDPD-WTUsing 1-palmitoyl-sn-3 phosphatidylcholine（16:0 LPC）as the substrate,³¹P-NMR was used to analyze the characteristics of the product formed during the hydrolysis of 16:0 LPC catalyzed by pfGDPD-WT.The results showed in addition to the lysophosphatidic acid（16:0LPA）production,the production of GPC was also found in the hydrolysate.The site-directed mutagenesis method was further used to construct mutants（H17A,H59A,H17A-H59A）targeting the critical catalytic residues His17,His59 in the active centre of pfGDPD.It was found that the product was GPC during the hydrolysis of 16:0 LPC catalyzed by the three mutants,and no LPA production was observed.The EDTA-2Na was used to chelate the metal ions which constituted the active center of the enzyme protein.The results showed that only GPC was observed in the hydrolysates of LPC hydrolyzed by pfGDPD.The above results indicated that the two histidines（His17 and His59）and metal ions,which constituted the active centre of the enzyme had nothing to do with the production of GPC.（2）Analysis of the crystal structure of pfGDPD-WTTo further clarify the molecular mechanism of GPC production in the process of the hydrolysis of 16:0 LPC catalyzed by pfGDPD,we tried to analyze the three-dimensional structure of its wild-type protein.The high-purity pfGDPD-WT protein was obtained by Ni²⁺-NTA affinity chromatography and gel filtration chromatography.By screening and optimization of crystallization conditions,pfGDPD-WT crystals were obtained.The diffraction data with a resolution of 1.65（?）was obtained by X-ray diffraction,then submitted to the PDB database（PDB:7E0V）.The pfGDPD monomer exhibits a TIM-barrel fold and displays a central eight-stranded parallelβ-sheet barrel,which is surrounded by nineα-helices.Its active catalytic centre is comprised of strictly conserved residues His17,Asp18,Glu44,Asp46,His59,Glu111,and Lys113,which form an electronegative cleft for binding substrates.（3）The effect of amino acids in the loop region of the active catalytic centre on the production of GPC in the process of enzymatic hydrolysis of LPCThrough analyzing the structure of pfGDPD-WT,two loops near the active catalytic centre,a series of sites on loop（60-63）and loop（67-72）were selected.The amino acids in the loop region affect the GPC production during the enzymatic hydrolysis of 16:0 LPC.Based on the pfGDPD-H17A-H59A double mutant,three-point mutations containing the corresponding sites were designed and constructed.As a result,no GPC was observed during the hydrolysis of 16:0 LPC catalyzed by pfGDPD-H17A-H59A-D60A.A single point mutation was further constructed based on pfGDPD-WT,and it was verified that the product of pfGDPD-D60A hydrolyzing 16:0 LPC was 16:0 LPA,and no GPC was observed during this process.This result indicated that D60 might be the critical amino acid that led to the production of GPC.（4）The crystal structure analysis of pfGDPD-D60A and D60A-16:0 LPC complexTo further understand why D60 site mutation causes GPC not to be produced by enzymatic hydrolysis of 16:0 LPC.We tried to analyze the mutant（PDB:7E2A）structure,and the crystal of D60A-16:0 LPC complex（PDB:7E2B）was obtained by enzyme-substrate co-incubation,with the resolution of 2.08（?）and 1.85（?）,respectively.By comparing with the structure of pfGDPD-WT,it was proposed that the mechanism of GPC production catalyzed by pfGDPD based on the critical amino acid D60.That was,D60 activated water molecules to cause nucleophilic attack on fatty acid ester bonds and breake them.