The Crystallographic Study Of The Enzymes Of PigD,PigB And PigE In The Biosynthetic Pathway Of Prodigiosin

Serratia spp could synthesize prodigiosin which has anti-bacterial,anti-fungal,anti-protozoal,anti-malaria,immunosuppressive and anti-cancer activities.The biosynthetic pathway of prodigiosin involve separate pathways for the production of the monopyrrole,2-methyl-3-n-amyl-pyrrole(MAP)and the bipyrrole,4-methoxy-2,2'-bipyrrole-5-carbaldehyde(MBC)which are then coupled in the final condensation step to produce prodigiosin.The MAP was biosynthesized by PigB,PigD and PigE.The gene of the PigE protein which catalized the 3-acetyloctanal to H2MAP in the MAP pathway was amplified by PCR using the genomic DNA of Serratia sp.FS14 as template,the PCR product was then cloned into the expression vector pET24b,the recombinated PigE was then expressed in E.coli C43(DE3).The puried PigE protein was then used for crystal screening,the best crystal we got was diffracted to 2.3A.The crystal structure of PigE was solved by molecular replacement method using 1VEF(acetyl ornithine aminotransferase)as the template.But the electron desity showed that the crystal only containing the catalytic domain of PigE,so we checked the diffracted crystal by SDS PAGE,the SDS gel verified that the crystal do only contain a 55 kDa protein,these results indicate the protein degradated during crystallization.The structure of PigE catalytic domain shows a homodimer in the asymmetric unit,each monomer is composed of three domains with PLP as cofactor.The residues from both monomers build the PLP binding sites at the interface of the dimer.Structural comparison of PigE with 1 VEF revealed a higher hydrophobic but smaller catalytic pocket,but which size is still big enough for the binding of its substrate 3-acetyloctanal.pigD and pigB genes were also amplified by PCR using the genomic DNA of Serratia sp.FS14 as template,and cloned into the expression vector for protein expression in E.coli C43(DE3).The puried PigD were also used for the crystal screening,but no crystals were obtained till now.Since the puried full-length membrane protein PigB was not stable,we expressed and the cytosolic portion of PigB protein for crystallization,but there is also no crystals obtained for the PigB-mowai protein even with extensive crystallization screen.