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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 v vlytB 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 VQ Q9/VN N2 T cells than isopentenyl pyrophosphate. ß

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