Hexamerization of geranylgeranylglyceryl phosphate synthase ensures structural integrity and catalytic activity at high temperatures.

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 degrees 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.