Archaebacterial ether-linked lipid biosynthetic gene. Expression cloning, sequencing, and characterization of geranylgeranyl-diphosphate synthase

Abstract

Archaebacterial Sulfolobus acidocaldarius geranylgeranyl-diphosphate (GGPP) synthase (EC 2.5.1.29) catalyzes consecutive condensations of isopentenyl diphosphate with allylic diphosphates to produce GGPP which is the important precursor of archaebacterial ether-linked lipids. We developed an expression screening method for cloning the GGPP synthase gene, which utilizes the carotenoid biosynthesis genes of Erwinia uredovora to visualize a clone expressing GGPP synthase, and then screened a genomic DNA library from S. acidocaldarius for the GGPP synthase gene by using this method. Positive clones were shown to contain GGPP synthase gene by the use of an in vitro assay. Extracts from Escherichia coli transformants catalyzed the condensation of isopentenyl diphosphate with farnesyl diphosphate (FPP) to produce (all-E)-GGPP. The nucleotide sequence of the 2.3-kilobase HindIII fragment of the cloned fragment was determined. This sequence specifies two open reading frames, ORF-1 and ORF-2. ORF-1 encodes GGPP synthase with the expected molecular weight of 36,873, and ORF-2 encodes a protein with homology for UDP-N-acetylglucosaminedolichyl phosphate N-acetylglucosaminephosphotransferase. The cloned GGPP synthase was partially purified with several chromatographies after heat treatment of cell free extract. This enzyme is extremely thermostable and has an optimal pH at 5.8. Dimethylallyl diphosphate, geranyl diphosphate, and (all-E)-FPP are, in decreasing order of activity, acceptable as allylic substrates to produce (all-E)-GGPP. When dimethylallyl diphosphate or geranyl diphosphate are the allylic substrates, a significant amount of mixture of the products is shorter than GGPP. (2Z,6E)-FPP is not a substrate. This enzyme recognizes the E-configuration of allylic substrate.