The structural gene for thermostable farnesyl diphosphate synthase from Bacillus stearothermophilus was cloned, sequenced, and overexpressed in Escherichia coli cells. A 1,260-nucleotide sequence of the cloned fragment was determined. This sequence specifies an open reading frame of 891 nucleotides for farnesyl diphosphate synthase. The deduced amino acid sequence shows a 42% similarity with that of E. coli FPP synthase [Fujisaki et al. (1990) J. Biochem. 108, 995-1000]. Comparison with prenyltransferases from a wide range of organisms, from bacteria to human, revealed the presence of seven highly conserved regions. In contrast to thermolabile prenyltransferases, which have four to six cysteine residues, the thermostable farnesyl diphosphate synthase carries only two cysteine residues. This enzyme is also unique in that some of the amino acids that are fully conserved in equivalents from other sources are replaced by functionally different amino acids. Construction of an overproducing strain provided a sufficient supply of this enzyme and it was purified to homogeneity. The purified recombinant enzyme is immunochemically identical with the native B. stearothermophilus enzyme, and it is not inactivated even after treatment at 65 degrees C for 70 min.

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The gene for thermostable farnesyl diphosphate synthase from Bacillus stearothermophilus was cloned, sequenced, and overexpressed in Escherichia coli. The synthase was purified to homogeneity and crystallized. The enzyme carried only two cysteine residues in contrast to its counterparts from other sources, which have four to six cysteine residues. Either or both of the cysteine residues can be replaced with serine without causing a loss of the catalytic activity. The conserved arginine residue that occupies the third position from the C-terminus was also replaced with valine without significant loss of activity, but the valine mutant showed a weakened affinity for isopentenyl diphosphate.