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Abstract
Cobamides (Cbas) are complex, cobalt-containing cyclic tetrapyrroles belonging to a family of cofactors called ‘the pigments of life.’ Complete synthesis of Cbas is restricted to bacteria and archaea, yet it is an essential cofactor for cells in all domains of life. Cbas are structurally unique from cofactors like hemes, chlorophylls and coenzyme F430 by their upper and lower ligands. Adenosylcobalamin (AdoCbl), the coenzymic form, is characterized by an upper 5’-deoxyadenosine moiety (Ado) and a 5,6-dimethylbenzimidazole (DMB) lower ligand. Prokaryotes utilize two distinct pathways for corrin ring synthesis: anaerobic or aerobic. These pathways converge at the synthesis and attachment of the lower ligand, a pathway referred to as Nucleotide Loop Assembly (NLA). NLA is characterized by two branches: i) the attachment of an aminopropanol-phosphate linker (CbiB EC 6.3.1.10) to and activation of the corrin ring (CobU EC 2.7.7.62/ EC 2.7.1.156) and ii) the activation of the nucleotide base (CobT EC 2.4.2.21). Cobamide 5’-phosphate synthase (CobS EC 2.7.8.26) joins these two pathways by condensing the activated corrin ring and nucleotide base substrates to form adenosylcobamide-phosphate (AdoCba-P). The final step of the pathway is catalyzed by CobC (EC 3.1.3.73) which dephosphorylates AboCba-P to form AdoCba. There are numerous homologous enzymes in bacteria and archaea responsible for carrying out the steps of NLA. Here we present a structural and functional characterization of MjCobT, an archaeal CobT homolog from hyperthermophile Methanocaldococcus jannaschii. Phylogenetic analysis of MjCobT uncovered the presence homologous base activating enzymes in cyanobacterial genomes, a group of organisms previously thought to lack CobT. In addition to our work on archaeal base activation, we sought to expand our understanding of CobS.
CobS is a polytopic integral membrane protein in all Cba producing organisms. This membrane conservation raises a number of questions about the significance of membrane-associated Cba biosynthesis. We developed a protocol for overproduction, purification and reconstitution of CobS into liposomes which led to the functional characterization of this enzyme. Additionally, we identified the physiological challenges excess CobS poses on the cell. Collectively, this work allowed us to begin unraveling the multi-protein complex of NLA enzymes, identifying CobS interactions with CobU, CobT and CobC.