The N-terminus and the chain-length determination domain play a role in the length of the isoprenoid product of the bifunctional Toxoplasma gondii farnesyl diphosphate synthase

Biochemistry. 2012 Sep 25;51(38):7533-40. doi: 10.1021/bi3005335. Epub 2012 Sep 12.

Abstract

Toxoplasma gondii possesses a bifunctional farnesyl diphosphate (FPP)/geranylgeranyl diphosphate (GGPP) synthase (TgFPPS) that synthesizes C(15) and C(20) isoprenoid diphosphates from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). This enzyme has a unique arrangement of the fourth and fifth amino acid upstream from the first aspartic rich motif (FARM) where the fourth amino acid is aromatic and the fifth is a cysteine. We mutated these amino acids, converting the enzyme to an absolute FPPS by changing the cysteine to a tyrosine. The enzyme could be converted to an absolute GGPPS by changing both the fourth and fifth amino acids to alanines. We also constructed four mutated TgFPPSs whose regions around the first aspartate rich motif were replaced with the corresponding regions of FPP synthases from Arabidopsis thaliana or Saccharomyces cerevisiae or with the corresponding regions of GGPP synthases from Homo sapiens or S. cerevisiae. We determined that the presence of a cysteine at the fifth position is essential for the TgFPPS bifunctionality. We also found that the length of the N-terminal domain plays a role in determining the specificity and the length of the isoprenoid product. Phylogenetic analysis supports the grouping of this enzyme with other type I FPPSs, but the biochemical data indicate that TgFPPS has unique characteristics that differentiate it from mammalian FPPSs and GGPPSs and is therefore an important drug target.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Chromatography, Thin Layer
  • Geranyltranstransferase / chemistry
  • Geranyltranstransferase / genetics
  • Geranyltranstransferase / metabolism*
  • Mutation
  • Phylogeny
  • Terpenes / chemistry*
  • Toxoplasma / enzymology*

Substances

  • Terpenes
  • Geranyltranstransferase