The Crystal Structure Analysis Of PigI And PigA From The Prodigiosin-Synthetic MBC Pathway

Prodiginines a typical secondary metabolite are a family of tripyrrole red pigments produced by actinomycetas, serratia marcescens and some bacteria, and have immunosuppressive, antibacterial, antifungal and antimalarial activities.The biosynthesis of prodigiosin was proposed with a bifurcated pathway, 4-methoxy-2,2’-bipyrrole-5-carbaldehyde (MBC) and 2-methyl-3-n-amyl-pyrrole (MAP) were separately synthesized before condensed to form prodigiosin by PigC. PigI is a putative L-prolyl-AMP ligase, which activates L-proline to L-prolyl-AMP and then tethers the L-prolyl-AMP to the phosphopantetheinylated form of PigG to form heterocyclic structure with the nitrogen atom. The L-prolyl-S-PigG is then desaturated to pyrrolyl-2-carboxyl- S- PigG by PigA, the 42 kDa flavoprotein desaturase.The enzymes involved in the biosynthetic pathways for prodigiosin are now well known. So far, prodiginine biosynthetic gene clusters have now been identified, sequenced and expressed in heterologous hosts, but the three-dimensional structures of these protein have not been reported. However, no structural and catalytic information are available for the biosythesis of prodigion. To elucidate the catalytic mechanism of PigI and PigA, the predicted genes coding for the PigI and PigA was amplified by PCR from a sample of Serratia sp. FS14, and then subcloned into the expression vector pET24b to generate the recombinant expression constructs, respectively. The PigI/PigA -encoding plasmid was transformed into E. coli C43 (DE3) cells for expression.The expression products were purified by Ni-NTA affinity chromatography and gel filtration chromatography. The protein samples were pure enough for crystallization screen. The initial crystallization conditions were then optimized. For data collection, the best crystal diffracted to a resolution of 1.8 A and 2.0 A respectively using synchrotron X-rays. Structures determination of Pigl was not successful with molecular replacement due to the low identity with its homologs. To solve the phase problem, the selenomethinine derivatized PigI crystals were prepared, and at the same time, we also cocrystallized PigI with heavy atoms. However, no obvious anomalous signal of Se atom and heavy atoms were detected.The structure of the PigA was solved by molecular replacement due to the high identity with its homologs. The monomeric structure of PigA is composed of three distinct domains: an N-terminal a-helical domain containing six helices (1-6), a medial P-sheet domain containing seven P-strands (1-7) and three short helices (7-9), and a C-terminal a-helical domain containing seven helices. The FAD cofactor adopts the relex conformation in the crystal structure, and tightly binds to the cleft through intensive hydrogen bonds and van der Waals interactions.