Structural and mechanistic insights into the biosynthesis of CDP-archaeol in membranes

Abstract

The divergence of archaea, bacteria and eukaryotes was a fundamental step in evolution. One marker of this event is a major difference in membrane lipid chemistry between these kingdoms. Whereas the membranes of bacteria and eukaryotes primarily consist of straight fatty acids ester-bonded to glycerol-3-phosphate, archaeal phospholipids consist of isoprenoid chains ether-bonded to glycerol-1-phosphate. Notably, the mechanisms underlying the biosynthesis of these lipids remain elusive. Here, we report the structure of the CDP-archaeol synthase (CarS) of Aeropyrum pernix (ApCarS) in the CTP- and Mg-bound state at a resolution of 2.4 Å. The enzyme comprises a transmembrane domain with five helices and cytoplasmic loops that together form a large charged cavity providing a binding site for CTP. Identification of the binding location of CTP and Mg enabled modeling of the specific lipophilic substrate-binding site, which was supported by site-directed mutagenesis, substrate-binding affinity analyses, and enzyme assays. We propose that archaeol binds within two hydrophobic membrane-embedded grooves formed by the flexible transmembrane helix 5 (TM5), together with TM1 and TM4. Collectively, structural comparisons and analyses, combined with functional studies, not only elucidated the mechanism governing the biosynthesis of phospholipids with ether-bonded isoprenoid chains by CTP transferase, but also provided insights into the evolution of this enzyme superfamily from archaea to bacteria and eukaryotes.

Structural and Functional Insights into an Archaeal Lipid Synthase

“…The two tunnels used by DGGGPase for substrate binding are similar to those of the CarS enzyme, the downstream enzyme of DGGGP in CDP-archaeol biosynthesis, and its structural homolog, TmCdsA (Liu et al, 2014), a bacterial CDP-DAG synthase (PDB: 4q2e and 4q2g). Both CarS and TmCdsA require bulky hydrophobic tunnels to accommodate substrates with two lipid tails (Ren et al, 2017), but they are structurally distinct. Overall, our findings reveal that members of the UbiA superfamily have distinct mechanisms of substrate recognition despite their similar structural motifs.…”

“…Membrane lipid strips were utilized as previously described (Dowler et al, 2002), and the detailed experimental procedures were as reported by Ren et al (2017). Briefly, 0.2 ml (0.005 mM) of synthesized lipids GGPP or GGGP was spotted onto Hybond-C extra membranes (GE Healthcare Life Sciences), and the membrane strips were dried using a hair dryer.…”

“…For example, the biosynthesis pathways for 2,3-di-O-geranylgeranylglyceryl phosphate (DGGGP) and 1-CDP-2,3-di-O-geranylgeranylglyceryl phosphate (CDParchaeol) are catalyzed by DGGGP synthase (DGGGPase) and CDP-archaeol synthase (CarS), respectively. The structure and function of CarS, the downstream enzyme of DGGGPase in this biosynthesis pathway, have recently been elucidated (Ren et al, 2017). However, the identity of DGGGPase is still elusive.…”

CDP-Diacylglycerol Synthases (CDS): Gateway to Phosphatidylinositol and Cardiolipin Synthesis

“…Nonetheless, the key step in phospholipid synthesis is the same in all three (Jain et al, 2014). The structure of the CarS of Aeropyrum pernix (ApCarS) comprises of five transmembrane helices and cytoplasmic loops which form a large charged cavity for CTP and the lipophilic substrate, DGGGP to bind (Ren et al, 2017). Part of the cavity is formed by two cytoplasmic loops, also contributing to substrate binding similar to TmCdsA (Figure 2A).…”

Membrane Lipid Composition and Amino Acid Excretion Patterns of Methanothermococcus okinawensis Grown in the Presence of Inhibitors Detected in the Enceladian Plume

“…Cell membranes of most terrestrial organisms are glycerol phosphate phospholipids [1]. In Bacteria and Eukarya, they occur in glycerol-3-phosphate configuration, while Archaea use glycerol-1-phosphate to build their polar head groups [2]. It was shown that precursors of lipids like glycerol-1-phosphate can be formed abiotically by prebiotic chemistry by reductive homologation of hydrogen cyanide and some of its derivatives [3].…”