Abstract 1983 Identifying the Role of the Rubredoxin-like Domain in Tetraether Lipid Synthesis

Archaeal membranes contain isoprenoid-based ether-linked lipids, which enable them to live in hostile conditions such as high temperature, high pressure, and high or low pH. In some archaeal extremophiles, such as Methanocaldococcus jannaschii, increased membrane stability is achieved through the synthesis of membrane-spanning tetraether lipids known as glycerol dibiphytanyl glycerol tetraether (GDGT). GDGT is formed by covalently linking the terminal carbons on the tails of two glycerophospholipids. In M. jannaschii, this reaction is catalyzed by the radical S-adenosylmethionine (SAM) enzyme GDGT-MAS. To catalyze the formation of this bond, GDGT-MAS couples two completely chemically inert sp3-hybridized carbons, which is the first time this reaction type has been observed in nature. GDGT-MAS contains four metallocofactors: three [Fe4S4] clusters and a rubredoxin-like iron. Currently, there have only been functions proposed for two of the iron-sulfur clusters (the radical SAM cluster and the C-terminal auxiliary cluster). In contrast, the roles of the third cluster (the N-terminal auxiliary cluster) and the rubredoxin-like iron remain unknown. Other examples of rubredoxin irons found throughout nature typically act as electron carriers. Therefore, we hypothesized that the rubredoxin-like iron plays a critical role in shuttling electrons into and out of the enzyme's active site. We used site-directed mutagenesis to create three mutants designed to remove the iron ion or to perturb the redox potential. We subsequently measured the catalytic impact of these mutations using established activity assays. Additionally, using X-ray crystallography, we plan to characterize the influence of this domain on protein structure. Our work aims to determine the catalytic and structural function of the rubredoxin domain during the synthesis of tetraether lipids by GDGT-MAS.