Structural And Functional Study Of Vibrio Vulnificus Phospholipase VvPlpA

Vibrio vulnificus is a mesophilic,halophilic,basophilic gram-negative marine pathogen that is widely distributed in estuaries and oceans.V.vulnificus can infect major economic fish through skin wounds or intestinal tracts.The infection is characterized by rapid onset and high mortality,and seriously endangers the development of aquaculture.The strong pathogenicity of V.vulnificus is directly related to its multiple virulence factors.The V.vulnificus phospholipase(VvPlpA)plays an important role in its pathogenicity.VvPlpA is secreted by the type II secretion system and catalyzes phosphatidylcholine as a specific substrate.The hydrolysate acts as a signaling molecule to stimulate the occurrence of inflammation,which in turn causes intense infection such as tissue necrosis and sepsis.Study on the virulence factor is conducive to understanding the pathogenic mechanisms of V.vulnificus.According to amino acid sequence alignment,VvPlpA has over 60%sequence similarity with homologs in vibrio,and belongs to the thermolabile hemolysin(TLH)family.But no structural information for this class of virulence factors is available.Sequence analysis showed VvPlpA has a typical sequence characteristic of the SGNH hydrolase superfamily.This superfamily is characterized by containing four conserved catalytic residues,serine,glycine,asparagine and histidine.Serine and histidine are the key catalytic residues,and usually cooperate with aspartate or glutamate preceding the histidine,thus forming a Ser-His-Asp/Glu catalytic triad.However,the aspartate/glutamate is substituted by a non-classical glycine in VvPlpA,and exhibits a non-canonical active site pattern.In this study,the recombinant protein VvPlpA was expressed in Escherichia coli.The crystal structure was solved at a high resolution by molecular replacement.The structure shows that the N-terminal domain mainly contains multiple?-sheets with unknown function,while the C-terminal domain has typical?/?/?structure characteristics and belongs to the SGNH hydrolase superfamily.The Ser152Gly,His392Asn and Asn247Asp mutants further verify the key role of the conserved residues in catalysis.Near the conserved His392 and atypical Gly389 of VvPlpA,we found a weak anomalous scattering signal and presumed it to be a chloride ion.Then the solved structure of bromine-substituted VvPlpA showed a strong anomalous scattering signal of bromine at the same position,indicating the substitution of bromine ion for chloride ion.The importance of Cl~-was tested by monitoring the chloride dependence of enzyme activity.The results showed that the activity of the wild type protein increases with increasing chloride ion concentration,while the activity of the Gly389Asp protein does not.It indicated that the chloride ion plays a key role in the catalytic function and forms an unusual Ser-His-chloride catalytic triad.At the same time,we analyzed the crystal structures of the Gly389Asp and Gly389Asn mutants which were used to simulate classical catalytic triad and dyad.We found that the chloride ion at the binding site was replaced by the side chain of each mutated residue.The structural characteristics were consistent with the performance of catalytic activity.A hexaethylene glycol was bound to the narrow pocket near the active site of the Gly389Asn mutant,providing information on substrate binding mode.