Thermostable farnesyl diphosphate synthase of Bacillus stearothermophilus: crystallization and site-directed mutagenesis.

Abstract

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.

Farnesyl diphosphate synthase; regulation of product specificity.

“…The Arg residue in the third position from the C-terminus of the enzyme (region VII) is highly conserved. Koyama et al (1994) examined the significance of this amino acid by replacing Arg295 in B. stearotermophilus with Val. This mutation, however, did not result in a marked change in catalytic activity, indicating that the Arg residue in region VII is not essential for catalysis (Koyama et al, 1994).…”

The biosynthesis of steroids and triterpenoids

“…1351 The FPS from B. stearothermophilus has less tolerance for oxygen-containing artificial substrates when compared with the eukaryotic enzyme. 1352 FPS from B. stearothermophilus 1353,1354 has been cloned. The mechanism by which prokaryotic squalene synthase converts FPP to SQ in Zymomonas mobilis may be different from that described for eukaryotes since in the absence of NADH an unidentified conversion product, not corresponding to presqualene alcohol, was isolated in good yield.…”

Isolation and characterization of a gene encoding farnesyl diphosphate synthase from \(\textit{Panax vietnamensis}\) Ha et Grushv

“…Farnesyl diphosphate synthase, or farnesyl pyrophosphate synthase, is a rate-limiting enzyme with a crucial role in one of the first steps in ginsenoside biosynthesis, catalyzing the condensation of isopentenyl diphosphate (IPP) with geranylgeranyl diphosphate (GPP) and dimethylallyl diphosphate (DMAPP) into farnesyl diphosphate (FPP) (Koyama et al, 1994). Despite abundant researches on gene FPS in P. ginseng and other species of genus Panax, up until now, there were no in-depth studies on this gene in P. vietnamensis.…”