RESUMO
The gcpE and lytB gene products control the terminal steps of isoprenoid biosynthesis via the 2-C-methyl-D-erythritol 4-phosphate pathway in Escherichia coli. In lytB-deficient mutants, a highly immunogenic compound accumulates significantly, compared to wild-type E. coli, but is apparently absent in gcpE-deficient mutants. Here, this compound was purified from E. coli DeltalytB mutants by preparative anion exchange chromatography, and identified by mass spectrometry, (1)H, (13)C and (31)P NMR spectroscopy, and NOESY analysis as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP). HMB-PP is 10(4) times more potent in activating human Vgamma9/Vdelta2 T cells than isopentenyl pyrophosphate.
Assuntos
Difosfatos/farmacologia , Enzimas , Eritritol/análogos & derivados , Proteínas de Escherichia coli , Escherichia coli/química , Ativação Linfocitária/efeitos dos fármacos , Mitógenos/farmacologia , Oxirredutases , Receptores de Antígenos de Linfócitos T gama-delta , Subpopulações de Linfócitos T/efeitos dos fármacos , Proteínas de Bactérias/genética , Difosfatos/química , Eritritol/biossíntese , Humanos , Mitógenos/química , Modelos Biológicos , Ressonância Magnética Nuclear Biomolecular , Espectrometria de Massas por Ionização por Electrospray , Fosfatos Açúcares/biossíntese , Terpenos/metabolismoRESUMO
The mevalonate-independent 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis is essential in many eubacteria, plants, and the malaria parasite. Using genetically engineered Escherichia coli cells able to utilize exogenously provided mevalonate for isoprenoid biosynthesis by the mevalonate pathway we demonstrate that the lytB gene is involved in the trunk line of the MEP pathway. Cells deleted for the essential lytB gene were viable only if the medium was supplemented with mevalonate or the cells were complemented with an episomal copy of lytB.
Assuntos
Proteínas de Bactérias/metabolismo , Eritritol/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Ácido Mevalônico/metabolismo , Oxirredutases , Fosfatos de Poli-Isoprenil/biossíntese , Fosfatos Açúcares/metabolismo , Animais , Proteínas de Bactérias/genética , Eritritol/análogos & derivados , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Deleção de Genes , Genes Bacterianos/genética , Genes Essenciais/genética , Teste de Complementação Genética , Humanos , Fosfatos de Poli-Isoprenil/metabolismo , Homologia de SequênciaRESUMO
In a variety of organisms, including plants and several eubacteria, isoprenoids are synthesized by the mevalonate-independent 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Although different enzymes of this pathway have been described, the terminal biosynthetic steps of the MEP pathway have not been fully elucidated. In this work, we demonstrate that the gcpE gene of Escherichia coli is involved in this pathway. E. coli cells were genetically engineered to utilize exogenously provided mevalonate for isoprenoid biosynthesis by the mevalonate pathway. These cells were then deleted for the essential gcpE gene and were viable only if the medium was supplemented with mevalonate or the cells were complemented with an episomal copy of gcpE.