Hexamerization and thermostability emerged very early during geranylgeranylglyceryl phosphate synthase evolution

Abstract

A large number of archaea live in hyperthermophilic environments. In consequence, their proteins need to adopt to these harsh conditions, including the enzymes that catalyze the synthesis of their membrane ether lipids. The enzyme that catalyzes the formation of the first ether bond in these lipids, geranylgeranylglyceryl phosphate synthase (GGGPS), exists as a hexamer in many hyperthermophilic archaea, and a recent study suggested that hexamerization serves for a fine‐tuning of the flexibility – stability trade‐off under hyperthermophilic conditions. We have recently reconstructed the sequences of ancestral group II GGGPS enzymes and now present a detailed biochemical characterization of nine of these predecessors, which allowed us to trace back the evolution of hexameric GGGPS and to draw conclusions about the properties of extant GGGPS branches that were not accessible to experiments up to now. Almost all ancestral GGGPS proteins formed hexamers, which demonstrates that hexamerization is even more widespread among the GGGPS family than previously assumed. Furthermore, all experimentally studied ancestral proteins showed high thermostability. Our results indicate that the hexameric oligomerization state and thermostability were present very early during the evolution of group II GGGPS, while the fine tuning of the flexibility – stability trade‐off developed very late, independent of the emergence of hexamerization.

Controlling Enzymatic Activity by Modulating the Oligomerization State via Chemical Rescue and Optical Control

“…We selected the GGGPS from Methanothermobacter thermautotrophicus (mtGGGPS) as model enzyme for our proof‐of‐concept study, which allowed us to base on previous results. [ 18 , 19b , 20 , 21 ] In the hexameric mtGGGPS, complex formation is mediated by three distinct interfaces: the dimer module interface, the interconnecting interface and the ring interface (Figure 1 B). The dimer module interface connects two monomers to a dimer.…”

“…We managed to hexamerize N4 by transplanting the ring interface from N12 into N4. The resulting variant AncGGGPS2_N4_IF_n12 (N4_IF_n12), which differs only in five contact interface residues from N4, [20] showed comparable activity as N12 (Figure S7E). Due to the inactivity of N4, N4_IF_n12 promised the possibility to establish an infinite switching factor by modulating its oligomerization state using a single mutation.…”

“…Cloning and site‐directed mutagenesis : All mutated variants as described in Table S1 were generated by QuickChange mutagenesis [25] with oligonucleotides listed in Table S2. For ONBY incorporation, a stop codon point mutation (TAG= amber ) was introduced into RGGGPS (previously called AncGGGPS2_N4_IF_n12 [20] ). To confirm the successful mutation, all genes were sequenced entirely.…”

“…To confirm the successful mutation, all genes were sequenced entirely. RGGGPS has been cloned previously, [20] as well as mtGGGPS. [19b] The sequence numbering for mtGGGPS used in this study refers to EMBL ENA entry AAB85058.…”

“…[19] Hexameric oligomerization is widely spread among this enzyme family (Figure 1 B, C), but can easily be disturbed by introducing mutations at the subunit interfaces. The hexamer then disassembles into dimers,[ 19b , 20 ] which is accompanied by a 380‐fold decrease in catalytic efficiency. [18] We made use of this effect and now disturbed the hexamer either by mutating an essential Lys in the hexamerization interface, which can be chemically rescued in the purified protein, or by inserting the UAA o ‐nitrobenzyl‐ O ‐tyrosine (ONBY) instead of an essential aromate, which can be optically decaged to a Tyr.…”