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Expression, crystallization and preliminary crystallographic data analysis of PigF, an O-methyltransferase from the prodigiosin-synthetic pathway in Serratia.

Liu, Shaowei; Ran, Tingting; Shen, Xiang; Xu, Langlai; Wang, Weiwu; Xu, Dongqing.
Acta Crystallogr Sect F Struct Biol Cryst Commun; 68(Pt 8): 898-901, 2012 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-22869117
Prodigiosin, which is a member of the prodiginines, is a red linear tripyrrole compound. A gene cluster for the biosynthesis of prodigiosin has been identified in Serratia and most genes in the cluster have been functionally assigned. A bifurcated biosynthetic pathway for prodigiosin has previously been determined. The last step in the biosynthetic pathway of 4-methoxy-2,2'-bipyrrole-5-carbaldehyde (MBC) is catalyzed by PigF, which transfers a methyl group to 4-hydroxy-2,2'-bipyrrole-5-carbaldehyde (HBC) to form the terminal product MBC, but its catalytic mechanism is not known. To elucidate its mechanism, recombinant PigF was purified and crystallized. The crystals belonged to space group P2(1), with unit-cell parameters a = 69.4, b = 52.4, c = 279.2 Å, ß = 96.8°. The native crystals may contain six molecules in the asymmetric unit, with a V(M) of 2.17 Å(3) Da(-1) and a solvent content of 43.43%. A full data set was collected at 2.6 Šresolution using synchrotron radiation on beamline BL17U of Shanghai Synchrotron Radiation Facility (SSRF), People's Republic of China. Molecular replacement was unsuccessful. To solve the structure of PigF by experimental phasing, selenomethionine-derivativized protein crystals were prepared from a condition with 0.01 M spermidine as an additive. One crystal diffracted to 1.9 Šresolution and a full data set was collected on beamline BL17U at SSRF. The crystal belonged to space group P2(1), with unit-cell parameters a = 69.0, b = 52.9, c = 93.4 Å, ß = 97.3°. Heavy-atom substructure determination and phasing by SAD clearly showed that the crystal contains two molecules in the asymmetric unit, with a V(M) of 2.19 Å(3) Da(-1) and a solvent content of 43.82%.
Selo DaSilva