Structural studies of N. maritimus acetyl-CoA/Propionyl-CoA carboxylase with X-Ray crystallography approach

The Archaea domain is among one of the most broadly distributed prokaryotic life forms on our planet. Most of the members of this domain are known to live in extreme environments and are involved in the fixation of inorganic compounds into organic compounds. Many ammonia-oxidizing archaea belonging to the phyla Crenarchaeota and Thaumarchaeota were found to assimilate inorganic carbon into organic carbon through a carbon fixation pathway called 3-hydroxypropionate/4-hydroxybutyrate (HP/HB) cycle. Interestingly, the marine species of the phylum Thaumarcheota were found to use a modified version of the HP/HB cycle. Among these organisms, the species N. maritimus was discovered to use the most energy-efficient carbon fixation mechanism under aerobic conditions. This feature provides N. maritimus several advantages to thrive in extreme oligotrophic environments. In the HP/HB cycle, the carboxylation of acetyl-CoA and propionyl-CoA is of great importance as its products are essential precursors for the maintenance of this cycle. Importantly, in N. maritimus, acetyl-CoA and propionyl-CoA are carboxylated by a biotin-dependent single bifunctional enzyme called Acetyl- CoA/Propionyl-CoA carboxylase. This enzyme is composed of three subunits: a carboxyltransferase Nmar_0272, carbamoyl phosphate synthase Nmar_0273, and a biotin-lipoyl attachment domain-containing protein Nmar_0274. Together, these subunits form a protein complex to become functional. In this study, we have purified and crystallized Nmar_0272 and Nmar_0274 subunits and determined the first ever structure of carboxyltransferase subunit Nmar_0272. The structure of Nmar_0272 exhibited an apo form dimer structure at 2.26 Å resolution and in P63 space group symmetry. Additionally, certain undetermined regions were reconstructed using homology and AI-based tools. Overall, our study presents a novel protein structure and serves as a starting point for elucidating the carbon fixation mechanisms in marine Thaumarchaeota.