The role of histidine-114 of Sulfolobus acidocaldarius geranylgeranyl diphosphate synthase in chain-length determination

AbstractSulfolobus acidocaldarius geranylgeranyl diphosphate synthase yields (all-E)-C20 prenyl diphosphate as a final product. The three-dimensional model of the enzyme suggested that removing two bulky residues at 77 and 114 would allow additional prenyl-chain elongation. To test this, we examined several mutants with substitutions at 77 and/or 114. As a result, the mutants, F77G, F77G and H114A, F77G and H114G, H114A, and H114G gave C30, C45, C50, C30 and C40 as the main long product, respectively. These observations indicate that histidine-114 plays a crucial role in chain-length determination along with phenylalanine-77

Archaeal Phospholipid Biosynthetic Pathway Reconstructed in Escherichia coli

A part of the biosynthetic pathway of archaeal membrane lipids, comprised of 4 archaeal enzymes, was reconstructed in the cells of Escherichia coli. The genes of the enzymes were cloned from a mesophilic methanogen, Methanosarcina acetivorans, and the activity of each enzyme was confirmed using recombinant proteins. In vitro radioassay showed that the 4 enzymes are sufficient to synthesize an intermediate of archaeal membrane lipid biosynthesis, that is, 2,3-di-O-geranylgeranyl-sn-glycerol-1-phosphate, from precursors that can be produced endogenously in E. coli. Introduction of the 4 genes into E. coli resulted in the production of archaeal-type lipids. Detailed liquid chromatography/electron spray ionization-mass spectrometry analyses showed that they are metabolites from the expected intermediate, that is, 2,3-di-O-geranylgeranyl-sn-glycerol and 2,3-di-O-geranylgeranyl-sn-glycerol-1-phosphoglycerol. The metabolic processes, that is, dephosphorylation and glycerol modification, are likely catalyzed by endogenous enzymes of E. coli

Specific Partial Reduction of Geranylgeranyl Diphosphate by an Enzyme from the Thermoacidophilic Archaeon Sulfolobus acidocaldarius Yields a Reactive Prenyl Donor, Not a Dead-End Product ▿

Geranylgeranyl reductase from Sulfolobus acidocaldarius was shown to catalyze the reduction of geranylgeranyl groups in the precursors of archaeal membrane lipids, generally reducing all four double bonds. However, when geranylgeranyl diphosphate was subjected to the reductase reaction, only three of the four double bonds were reduced. Mass spectrometry and acid hydrolysis indicated that the allylic double bond was preserved in the partially reduced product derived from geranylgeranyl diphosphate. Thus, the reaction product was shown to be phytyl diphosphate, which is a substrate for archaeal prenyltransferases, unlike the completely reduced compound phytanyl diphosphate