Structural And Functional Studies On DUTP Pyrophosphatases From White Spot Syndrome Virus And Litopenaeus Vannamei

WSSV(white spot syndrome virus)is an important pathogen for crustaceans all over the world and has not been effectively controlled so far.The development of antiviral drugs is of great significance for the healthy and sustainable development of the crustacean culture industry.WSSV genome encodes dUTPase(dUTP Pyrophosphatase),a key enzyme in the thymidylate biosynthesis pathway.dUTPases that exist in most organisms,can catalyze the hydrolysis of dUTP to dUMP and pyrophosphate PPi,playing essential roles for DNA synthesis and stability.It is an important target for the development of anti-pathogenic drugs.The development of anti-WSSV drugs targeting to its dUTPase requires detailed understanding of the structure characteristics of WSSV and host dUTPases.In the present study,structural and functional characteristics of the conserved dUTPase domains from WSSV and Litopenaeus vannamei,an important culture species infected by WSSV,were studied by crystal structure analysis,residue site-directed mutagenesis,enzyme kinetic analysis and other methods.The results enriched the current knowledge of the classification and catalytic reaction mechanism of the dUTPase family and provided the accurate protein structural basis for the development of anti-WSSV drugs.The amino acid sequence alignment showed that,the N-terminal 171 residues of the wsv112-encoded protein(wDUT)in the WSSV belongs to the trimeric dUTPase family with five conserved motifs(I-V)which are necessary for the enzyme catalysis.Interstingly,wDUT harbors a unusual 20-residue(Tyr134-Asn153)insert(pre-V)at the position preceding the C-terminal catalytic motif V.wDUT crystal structure analysis revealed that the insert pre-V forms an unusual β-hairpin structure in the domain-swapping region and thereby facilitates a unique orientation of the adjacent C-terminal segment,bringing the catalytic motif V onto the active site of its own subunit.As a result,wDUT forms a new subclass of the homotrimeric dUTPase family with the noncanonical two-subunit active site assembly,distinct from the classic three-subunit active site assembly.This finding expands the current classification knowledge of the dUTPase family.The residues at the active site and the catalytic mechanism of wDUT are basically the same as the classic trimeric dUTPase through structure comparison and enzyme kinetic analysis of site-directed mutants.However,deletion of the pre-V insert significantly reduces wDUT’s enzymatic activity and thermal stability.This result suggested that the classic three-subunit active site in wDUT without the pre-V insert lost the high catalytic power,implying that wDUT has residue substitutions adapting to its novel two-subunit active site.Through the enzymatic kinetic analysis of wDUT and Escherichia coli dUTPase(prototype model in trimeric family)mutants,a conserved arginine/lysine located in motif Ⅳ of the classic dUTPases was proved to be functionally substituted by an unusual arginine(residue Arg24)at the position preceding motif I in wDUT.This rearrangement of wDUT residues occurs in the second shell of the active site is compatible with its novel C-terminal orientation to maintains the high catalytic power,suggesting that the evolution of wDUT should be considered the combined result of sequence insertion and amino acid substitution.The amino acid sequence alignment and crystal structure comparison showed that,the C-terminal 146 residues of dUTPase from L.vannamei(LvDUT)constitute a traditional trimeric dUTPase domain,distinct from the wDUT of WSSV.The comparison of LvDUT crystal structures in various reaction stages indicated that,dUTP binding can induce two kinds of novel conformational changes in the main body of dUTPases:the loop-to-alpha helix conformational change of the Ser132-His138 in the active site region and the "open-close" change of the trimer central channel.These conformational changes together promoted the reaction of dUTP hydrolysis.The structure comparison between wDUT and LvDUT showed that,both share essentially identical structures in two traditional sites of trimeric dUTPases for inhibitor design,including the substrate binding pocket and the trimer central channel,making it difficult to develop and use specific inhibitors targeting to wDUT.However,an appropriate small-molecule binding site is found in the space between the Lys150-Asn155 region and the mainbody in wDUT and can serve as a potential antiviral target for the wDUT inhibitor design.This finding provides the structural basis for developing specific antiviral drugs against WSSV infections.