Purification and characterization of geranylgeranylglyceryl phosphate synthase from a thermoacidophilic archaeon, Thermoplasma acidophilum

J Biochem. 2003 May;133(5):651-7. doi: 10.1093/jb/mvg083.

Abstract

We purified a geranylgeranylglyceryl phosphate (GGGP) synthase from Thermoplasma acidophilum by several steps of chromatography. Based on the proteinase-fragment-mass-pattern analysis of the SDS-PAGE band of the partially purified protein, the DNA sequence encoding the protein was identified from the whole genome sequence database of the species. The gene encoding GGGP synthase in T. acidophilum was cloned after PCR amplification of the gene from the genomic DNA. The recombinant enzyme was expressed in Escherichia coli and purified. A single band with a molecular mass of 27 kDa was obtained by SDS-PAGE analysis. The apparent native molecular mass of the enzyme was about 50 kDa based on gel filtration chromatography, suggesting that the enzyme is active as a homodimer. As the GGGP synthase from Methanobacterium thermoautotrophicum has been reported as a pentamer, the enzymes of the two organisms have different oligomeric structures. Other characteristics, including substrate specificity, are similar for the GGGPs of these organisms.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkyl and Aryl Transferases / chemistry
  • Alkyl and Aryl Transferases / genetics
  • Alkyl and Aryl Transferases / isolation & purification*
  • Alkyl and Aryl Transferases / metabolism*
  • Amino Acid Sequence
  • Archaeal Proteins / chemistry
  • Archaeal Proteins / genetics
  • Archaeal Proteins / isolation & purification*
  • Archaeal Proteins / metabolism
  • Dithiothreitol / pharmacology
  • Edetic Acid / pharmacology
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Glycerophosphates / chemistry
  • Glycerophosphates / metabolism
  • Hydrogen-Ion Concentration
  • Molecular Sequence Data
  • Molecular Weight
  • Octoxynol / pharmacology
  • Phylogeny
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Substrate Specificity
  • Temperature
  • Thermoplasma / enzymology*
  • Thermoplasma / metabolism

Substances

  • Archaeal Proteins
  • Glycerophosphates
  • Recombinant Proteins
  • Octoxynol
  • Edetic Acid
  • alpha-glycerophosphoric acid
  • Alkyl and Aryl Transferases
  • phosphoglycerol geranylgeranyltransferase
  • Dithiothreitol