Crystal Structure And Function Analysis Of PigD And PigE,Two Essential Enzymes Involved In Prodigisin-Biosynthesis

Prodigiosin,featured with tripyrrole ring,is a red-pigmented secondary metabolite synthesized by Serrratia,Pseudoalteromonas,some actinomycetes and other bacterias.Prodigiosin has long been a promising drug due to its anti-cancer,antimalarial,antibacterial,antifungal and immunosuppressive activities.The encoded enzymes of pig gene cluster(pigA-N)have been reported to be involved in the biosynthesis of prodigiosin.Studies have also revealed that prodigiosin is biosynthesized by an independent bifurcated pathway to synthesize two intermediate products 2-methyl-3-n-amylpyrrole(MAP)and 4-methoxy-2,2-bipyrrole-5-carboxyaldehyde(MBC),and the terminal condensing enzyme PigC finally binds MBC and MAP together to form prodigiosin.MAP in Serratia is reported to be synthesized by PigB,PigD and PigE.In order to further clarify the mechanism of PigD and PigE involved in the synthetic pathway of MAP,in this study,genes coding for PigD and PigE were cloned into the expression vector pColdX2,and the recombinant expression plasmids were then transformed into the host S.marcescens FS14 for homologous expression.PigD and PigE were then purified and analyzed through Ni-NTA affinity chromatography and gel filtration.It was found that the homologously expressed PigD is poor in purity and stability.Though PigE is in good purity and is homogenous,however,it is poor in stability either.Crystal screening was also performed,but,no crystal was observed so far.A large amount of stable and high-purity PigD proteins heterologously expressed in E.coli were then obtained by optimizing the former purification procedures.The crystallization screening of the PigD protein with the addition of substrate 2-Octenal and coenzyme TPP was then carried out.Finally,crystals of PigD were obtained from condition 20℃,0.1M HEPES pH 7.4,7%PEG 8000,and 8%Ethylene glycol.A full differaction data-set was collected to 2.7 ? resolution.As no homologous protein structure of PigD has been reported,we failed to solve the crystal structure of PigD by molecular replacement method.Therefore,the selenomethionine derived PigD protein was prepared for crystal screening.Unfortunately,no crystals of selenomethionine derived PigD has been obtained up to now.We also attempted to soak PigD protein crystals with selenourea,but the crystals were not stable when exposed to selenourea.Though the structure of the C-terminal domain(372-853 aa)of PigE has been resolved and its catalytic function has been clarified,the importance of the N-terminal(1-371 aa)domain of PigE is still elusive.In order to identify the importance of the N-terminal domain of PigE in S.marcescens FS14,in this report,the N-terminal deletion mutant of pigE(FS14ΔpigE2-371)and the full-length pigE deletion mutant(FS14ΔpigE)were constructed respectively,the results showed that both mutants could not synthesize prodigiosin.The complementary assay confirmed that PigE or the N-terminal domain of PigE are essential for prodigiosin synthesis.