Geranylgeranylglyceryl phosphate synthase.: Characterization of the recombinant enzyme from Methanobacterium thermoautotrophicum

Geranylgeranylglyceryl diphosphate synthase (GGGP synthase) catalyzes alkylation of (S)glyceryl phosphate [(S)-GP] by geranylgeranyl diphosphate (GGPP) to produce (S)-geranylgeranylglyceryl phosphate [(S)-GGGP]. This reaction is the first committed step in the biosynthesis of ether-linked membrane lipids in Archaea. The gene encoding GGGP synthase from Methanobacterium thermoautotrophicum was cloned using probes designed from the N-terminal sequence determined from the purified enzyme. The open reading frame, which encoded a protein of 245 amino acids, was inserted into a pET expression vector and expressed in Escherichia coli. The recombinant GGGP synthase was purified to homogeneity. The enzyme is active as a homopentamer, as determined by size exclusion chromatography and equilibrium sedimentation experiments. GGGP synthase has optimal activity at 55 degreesC in pH 8.0 buffer containing 1 MM MgCl2. V-max = 4.0 +/-0.1 mu mol min(-1) mg(-1) (k(cat) = 0.34 +/-0.03 s(-1) for pentameric GGGP synthase assuming all subunits are fully active), K-m((S)-GP) = 13.5 +/-1.0 muM, and K-m(GGPP) = 506 +/- 47 nM. These steady-state catalytic constants were identical to those for enzyme isolated from cell extracts of M. thermoautotrophicum [Chen, A., Zhang, D., and Poulter, C. D. (1993) J. Biol. Chem. 268, 21701-21705]. Alignment of seven putative archaeal GGGP synthase sequences revealed a number of highly conserved residues consisting of five aspartate/glutamates, three serine/threonines, two prolines, and five glycines, including a conserved GGG motif.