Structural studies of geranylgeranylglyceryl phosphate synthase, a prenyltransferase found in thermophilic Euryarchaeota

Abstract

Archaea are uniquely adapted to thrive in harsh environments, and one of these adaptations involves the archaeal membrane lipids, which are characterized by their isoprenoid alkyl chains connected via ether linkages to glycerol 1-phosphate. The membrane lipids of the thermophilic and acidophilic euryarchaeota Thermoplasma volcanium are exclusively glycerol dibiphytanyl glycerol tetraethers. The first committed step in the biosynthetic pathway of these archaeal lipids is the formation of the ether linkage between glycerol 1-phosphate and geranylgeranyl diphosphate, and is catalyzed by the enzyme geranylgeranylglyceryl phosphate synthase (GGGPS). The 1.72 Å resolution crystal structure of GGGPS from T. volcanium (TvGGGPS) in complex with glycerol and sulfate is reported here. The crystal structure reveals TvGGGPS to be a dimer, which is consistent with the absence of the aromatic anchor residue in helix α5a that is required for hexamerization in other GGGPS homologs; the hexameric quaternary structure in GGGPS is thought to provide thermostability. A phylogenetic analysis of the Euryarchaeota and a parallel ancestral state reconstruction investigated the relationship between optimal growth temperature and the ancestral sequences. The presence of an aromatic anchor residue is not explained by temperature as an ecological parameter. An examination of the active site of the TvGGGPS dimer revealed that it may be able to accommodate longer isoprenoid substrates, supporting an alternative pathway of isoprenoid membrane-lipid synthesis.

Keywords: Thermoplasma volcanium; ancestral state reconstruction; archaea; ether lipids; geranylgeranyglyceryl phosphate synthase; isoprenoids; volcanic.

Figure 1

(a) Modified mevalonic acid pathway for the synthesis of IPP in extreme acidophiles of the order Thermoplasmatales, which includes T. volcanium. (b) GGPPS catalyzes the condensation of DMAPP with three units of IPP to form geranylgeranyl pyrophosphate. The first committed step in the biosynthesis of archaeal membrane lipids is the formation of the ether linkage between GGPP and glycerol 1-phosphate. (c) The proposed mechanism for ether-bond formation involves ionization of GGPP to form the allylic carbocation, followed by nucleophilic attack of the C3 OH group of glycerol at C1 of the geranylgeranyl carbocation. Glu172 of TvGGGPS acts as a base to activate glycerol for nucleophilic attack.

Figure 2

The phylogenetic reconstruction used to estimate ancestral GGGPS sequences and ancestral growth-temperature optima. (a) Phylogram showing the maximum-likelihood reconstruction of the relationships between the GGGPS amino-acid sequences from selected members of the Euryarchaeota. See Section 3 for the phylogenetic reconstruction details. The colored nodes represent aBayes support values (see scale). The nodes used for the alignment in Fig. 7 ▸ are labeled. (b) A ladderized version of the tree in (a) showing optimal growth-temperature changes throughout GGGPS evolution. The parallel bars at the base of the tree represent branches that were condensed to easily visualize the tree. Species names are highlighted based on the amino acid inhabiting the ‘anchor’ position in the GGGPS protein. W, tryptophan; Y, tyrosine; F, phenylalanine.

Figure 3

Stereoviews of the TvGGGPS active site. A mixture of cartoon-style ribbon plots and side-chain stick styles depict the active site, with C atoms in cyan, S atoms in yellow, N atoms in blue, O atoms in red, H₂O molecules as red spheres and glycerol C atoms in purple. Top: simulated-annealing mF _o − DF _c maps (green) contoured at 3σ show a well ordered placement of sulfate and glycerol ligands. Bottom: hydrogen-bonding interactions (blue dashed lines) reveal ligand-binding interactions and the water network (red translucent spheres). The sulfate and glycerol form hydrogen bonds to β6 (Tyr170 and Glu172), loop β6–α6 (Ser175 and Gly176) and loop β7–α7 (Gly202 and Arg204). While sulfate and glycerol are bound, the active site of TvGGGPS appears to only be partially closed since Gly223 and Ser224 from loop β8–α8 of TvGGGPS do not interact with either ligand in the active site.

Figure 4

Loop β3–α3* forms a random coil that extends into the isoprenoid-binding site of TvGGGPS, acting as a gatekeeper for isoprenoid binding. (a) The active-site surface cavity is highlighted for TvGGGPS, PcrB and AfGGGPS and their respective bound ligands displayed as cartoons. The red arrow is used as a frame of reference between each structure, highlighting the top of the α-helix in PcrB and the relative position of the random coil in TvGGGPS and AfGGGPS. (b) FSPP bound in PcrB is overlaid in the TvGGGPS and AfGGGPS isoprenoid-binding sites, emphasizing the mobility of the random coil in isoprenoid binding. In TvGGGPS, the random coil (Pro78–Ser81) must relocate to bind the 20-carbon GGPP. This may occur by forming a helix similar to the helix in PcrB or by moving the random coil outwards similar to the positioning of the random coil in AfGGGPS.

Figure 5

Hexamerization of GGGPS blocks one end of the isoprenoid-binding site. (a) The TvGGGPS dimer is displayed as the overall surface with one monomer in light gray and one monomer in dark gray. The isoprenoid active site is highlighted in blue (entrance) and red (exit). (b) Hexamer formation appears to block the proposed exit (red) of the isoprenoid channel. The other dimers from MtGGGPS (PDB entry 4mm1; pink and green surface) are superimposed onto the TvGGGPS dimer, showing the complete obstruction of the potential surface-accessible active-site exit upon hexamer formation.

Figure 6

A box and whisker plot showing the relationship between growth-temperature optima and the amino acid occupying the ‘anchor’ position for oligomerization. W, tryptophan; Y, tyrosine; F, phenylalanine; Other, any other amino acid. GGGPS homologs with a tryptophan at the anchor position had a significantly lower growth-temperature optimum than those with a Y, F or ‘Other’ at this position (p = 0.04195, p = 0.00898 and p = 0.00101, respectively). Comparisons of the growth-temperature optima for those with a Y, F or ‘Other’ at this position were not significant. Statistical analyses were performed by using an ANOVA test with a post hoc Tukey honest significance test. See Supplementary Table S3 for statistical details. *, p < 0.05; **, p < 0.01. Center lines show the medians, box limits indicate the 25th and 75th percentiles as determined by the R software, whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles and outliers are represented by dots.

Figure 7

Alignment of the ancestral state reconstruction of the GGGPS amino-acid sequences of ancestral nodes leading to the extant TvGGGPS and MtGGGPS sequences. See Fig. 2 ▸ for node positions. Residues that are likely to coordinate to magnesium ions are indicated with an asterisk. Residues that are likely to hydrogen-bond directly to the phosphate moiety of G1P are indicated with a dagger. Residues that are likely to hydrogen-bond directly to the glyceryl hydroxyls of G1P are indicated with a double dagger. The aromatic anchor residue position is indicated with a filled circle.