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

Abstract

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.

FIG. 1.

Biosynthesis of membrane lipid and caldariellaquinone in Sulfolobus sp. Each dashed arrow indicates a biosynthetic route catalyzed by multiple enzymes. In Sulfolobus sp., GGPP is thought to be the common precursor of hydrophobic isoprenoid compounds such as archaeal membrane lipid and caldariellaquinone (8, 11). In the largest box, two hypothetical products from GGPP obtained from the reaction with GGR from S. acidocaldarius are shown. Phytyl diphosphate, which is the final product of plant GGR, may be utilized by archaeal prenyltransferases for the biosynthesis of the isoprenoid compounds, while phytanyl diphosphate must be a dead-end product because it cannot be a prenyl donor. 3 × IPP, three molecules of IPP.

FIG. 2.

Mass spectrum of the product from the GGR reaction with DGGGP. DGGGP was reduced with an excess amount of S. acidocaldarius GGR, and the product was treated with phosphatase. The resulting alcohol, trimethylsylilated before the GC-MS analysis, gave a peak with the same GC retention time as the peak of authentic trimethylsylilated archaeol. The ion spectrum of the peak was similar to that of the authentic molecule. The ions at m/z 710, 621, and 426 correspond to [M-CH₃]⁺, [M-CH₃OSi(CH₃)₃]⁺, and [M-C₂₀H₄₁OH]⁺, respectively.

FIG. 3.

Radio-TLC analyses of the products from the GGR reactions with GGGP (A) and GGPP (B). After hydrolysis with phosphatase, the compounds were analyzed by reversed-phase TLC developed with acetone/H₂O (9:1). Lane 1, phosphatase-treated product from the reaction with S. acidocaldarius GGR; lane 2, phosphatase-treated product from the reaction with A. fulgidus GGR; lane 3, authentic geranylgeranylglycerol (A) and geranylgeraniol (B). s.f., solvent front; ori., origin.

FIG. 4.

Mass spectrum of the product from the GGR reaction with GGPP. GGPP was reduced with an excess amount of S. acidocaldarius GGR, and the product was treated with phosphatase. The retention time of the GC peak of the resulting alcohol corresponded with that of one of the peaks of authentic phytol isomers. The ion spectrum of the peak was similar to those of the authentic compounds. The ions at m/z 296 and 278 correspond to M⁺ and [M-H₂O]⁺, respectively.

FIG. 5.

Hydrolysis of the GGR reaction product from GGPP. Radiolabeled GGPP and GGPP reduced with S. acidocaldarius GGR were hydrolyzed by acid or phosphatase treatment and then analyzed by normal-phase TLC developed with 2-propanol-aqueous NH₄OH-H₂O (6/3/1). Lane 1, untreated GGPP; lane 2, acid-treated GGPP; lane 3, phosphatase-treated GGPP; lane 4, untreated GGR product from GGPP; lane 5, acid-treated GGR product from GGPP; lane 6, phosphatase-treated GGR product from GGPP. s.f., solvent front; ori., origin.

FIG. 6.

Determination of the substrate preferences of S. solfataricus prenyltransferases. (A) Radiolabeled phytyl diphosphate (lane 1) or GGPP (lane 2) was used as a substrate for a GGGP synthase reaction with racemic glyceryl-1-phosphate. After the reaction, radiolabeled compounds were extracted with butanol and then treated with phosphatase. The resulting alcohols were analyzed by reversed-phase TLC developed with acetone/H₂O (9:1). s.f., solvent front; ori., origin. (B) Nonlabeled phytyl diphosphate (lane 1) or GGPP (lane 2) was used as a substrate for a HexPP synthase reaction with [¹⁴C]IPP. After the reaction, radiolabeled compounds were extracted with butanol and then treated with phosphatase. The resulting alcohols were analyzed by reversed-phase TLC developed with acetone/H₂O (19:1).