Identification of radical SAM enzymes responsible for the methylation and desaturation of archaeal lipids and an AttH hydratase mediating hydroxy-GDGT biosynthesis

Lipid biosynthesis in archaea is highly dependent upon radical ( S )-adenosyl methionine (rSAM) enzymes. The formation of their membrane-spanning lipids, known as glycerol dibiphytanyl glycerol tetraethers (GDGTs), is catalyzed by the rSAM tetraether synthase and the subsequent cyclization of their lipid tails is performed by the B12-binding rSAM (B12-rSAM) GDGT ring synthase. Further, GDGT tails are cross-linked by the rSAM GMGT synthase and then methylated by the B12-rSAM GMGT methylase. Here, utilizing three archaeal model organisms – Sulfolobus acidocaldarius , Thermococcus kodakarensis , and Methanosarcina acetivorans – we expand this repertoire of rSAM enzymes further, identifying two B12-rSAM GDGT methylases and a novel B12-rSAM GDGT desaturase. We also extend beyond the rSAM enzyme superfamily, identifying an AttH-like hydratase mediating hydroxy-GDGT (OH-GDGT) biosynthesis. Specifically, we identify two GDGT methylases in S. acidocaldarius and Thermococcus aggregans which possess different substrate specificities. While GDGT methylation is generally low in archaea, we observe high levels of lipid methylation in response to penicillin G and hexanoic acid amphiphile exposure, suggesting a role for lipid methylation in response to membrane-destabilizing chemical agents. Utilizing heterologous expression in M. acetivorans and T. kodakarensis , we uncover a novel B12-rSAM enzyme from Candidatus Bathyarchaeota B1_G15 with unusual tetraether desaturase (Ted) activity that seemingly reverses previous saturation by geranylgeranyl reductase, anaerobically forming double bonds in the inert hydrocarbon tails of a GDGT. Finally, we show that a non-rSAM AttH-like hydratase, genomically associated with Ted, is a hydroxy-GDGT synthase (Hgs), putatively hydrating the double bond(s) introduced by Ted to form OH-GDGTs. These results highlight the value of exploring the rSAM enzyme landscape of archaea – revealing diverse and novel rSAM enzyme activities amongst homologous proteins that invoke new paradigms such as reversibility in the archaeal lipid biosynthesis pathway.