Hexamerization of Geranylgeranylglyceryl Phosphate Synthase Ensures Structural Integrity and Catalytic Activity at High Temperatures

Abstract

The cell membranes of all archaea contain ether lipids, and a number of archaea are hyperthermophilic. Consequently, the enzymes that catalyze the synthesis of membrane ether lipids had to adopt to these rough conditions. Interestingly, the enzyme that establishes the first ether bond in these lipids, the geranylgeranylglyceryl phosphate synthase (GGGPS), forms hexamers in many hyperthermophilic archaea, while also dimeric variants of this enzyme exist in other species. We used Methanothermobacter thermautotrophicus GGGPS (mtGGGPS) as a model to elucidate the benefit of hexamerization. We studied the oligomerization interfaces in detail by introducing disturbing mutations and subsequently compared the stability and activity of generated dimeric and monomeric variants with the wild-type enzyme. Differential scanning calorimetry revealed a biphasic denaturation of mtGGGPS. The temperature of the first transition varies and rises with increasing oligomerization state. This first phase of denaturation leads to catalytic inactivation, but CD spectroscopy indicated only minor changes on the secondary structure level. The residual part of the fold is extremely thermostable and denatures in a second phase at temperatures >120 °C. The analysis of another distant native GGGPS enzyme affirms these observations. Molecular dynamics simulations revealed three structural elements close to the substrate binding sites with elevated flexibility. We assume that hexamerization might stabilize these structures, and kinetic studies support this hypothesis for the binding pocket of the substrate glycerol 1-phosphate. Oligomerization might thus positively affect the thermostability-flexibility trade-off in GGGPS by allowing a higher intrinsic flexibility of the individual protomers.

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

“…Interestingly, the crystal structure of MtGGGPS revealed a hexamer that can be described as a trimer of dimers. Several other group II archaeal GGGPS enzymes have been identified as forming hexamers via static light-scattering experiments (Peterhoff et al, 2014;Linde et al, 2018). The quaternary structure of the MtGGGPS dimer is identical to that of the TvGGGPS dimer.…”

“…Mutagenesis experiments revealed that replacing Trp141 in MtGGGPS with an alanine residue results in disruption of the hexamerization interface, favoring formation of the dimer. Furthermore, differential scanning calorimetry and fluorimetry experiments indicate that the MtGGGPS hexamer has a significantly higher thermostability relative to the dimer and monomer (Linde et al, 2018). Oligomerization is a common adaptation of thermophilic proteins, along with an increased number of disulfide bonds, increased salt-bridging and increased surface charges (Reed et al, 2013).…”

“…Oligomerization is a common adaptation of thermophilic proteins, along with an increased number of disulfide bonds, increased salt-bridging and increased surface charges (Reed et al, 2013). Indeed, an in vitro mutagenic analysis by Linde et al (2018) led to the conclusion that the hexamerization of the M. thermautotrophicus GGGPS homolog ensures its structural and functional stability at the high growth-temperature optimum (65-70 C). Additionally, using protein sequencesimilarity analyses, Peterhoff et al (2014) suggested that an aromatic amino-acid anchor (tryptophan, tyrosine or phenylalanine) is necessary for the hexamerization of GGGPS proteins.…”

“…Homologs of GGGPS adopt different oligomerization states: FjGGGPS and AfGGGPS are dimers, while MtGGGPS is a hexamer. The hexamerization state of MtGGGPS is thought to provide thermostability in hyperthermophilic organisms (Linde et al, 2018).…”

Sequence selection by FitSS4ASR alleviates ancestral sequence reconstruction as exemplified for geranylgeranylglyceryl phosphate synthase

“…This enzyme is involved in the biosynthesis of ether membrane lipids that are prototypical for Archaea and catalyzes the formation of an ether bond between glycerol 1-phosphate and geranylgeranyl diphosphate (Chen et al, 1993;Peterhoff et al, 2014). A characteristic, taxonspecific property of GGGPS is the oligomerization state, which can be dimeric or hexameric (Peterhoff et al, 2014;Linde et al, 2018). We used the same ASR protocol, but two different sets of recent GGGPS homologs to compute ancestors.…”