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 degrees C in pH 8.0 buffer containing 1 mM MgCl(2). V(max) = 4.0 +/- 0.1 micromol 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 microM, 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.
Soderberg
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Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, USA.
Chen
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Poulter
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D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017014:Methanobacterium / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D021241:Spectrometry, Mass, Electrospray Ionization
D019843:Archaeal Proteins; D011994:Recombinant Proteins; D019883:Alkyl and Aryl Transferases; C083737:phosphoglycerol geranylgeranyltransferase