Biochemical Characterization of the GTP:Adenosylcobinamide-phosphate Guanylyltransferase (CobY) Enzyme of the Hyperthermophilic Archaeon Methanocaldococcus jannaschii

Cobamides are ancient cofactors that are widely distributed in nature (1). With the exception of plants, cells of all domains of life have enzymes that require a cobamide as their coenzyme, yet only some bacteria and archaea synthesize cobamides de novo (2–4). Although not all cobamide producers synthesize the corrin ring de novo, some can salvage pre-formed, incomplete precursors [e.g., cobinamide (Cbi), cobyric acid (Cby)] from their environments using a high- affinity ATP-binding cassette transporter (5–10). Precursors such as Cbi and Cby are then converted to cobamides by two distinct branches of the pathway. One of these branches, known as the corrinoid adenosylation pathway, attaches the upper axial ligand to the corrin ring via a labile C-Co bond (11). The second branch assembles the nucleotide loop that tethers the lower ligand base to the corrin ring; this branch is known as the nucleotide loop assembly pathway (Fig. 1). The latter can be further broken down into two sub-branches, one of which activates adenosyl-Cbi (AdoCbi) to AdoCbi-guanosine diphosphate (AdoCbi-GDP) (12), and a second one that activates the lower ligand base to its nucleotide (2) (Fig. 1). Figure 1 Nucleotide loop assembly pathway In bacteria, the activation of AdoCbi to AdoCbi-GDP is catalyzed by the bifunctional CobU enzyme (EC 2.7.1.156, EC 2.7.7.62) via an AdoCbi-P intermediate (13). Archaea, however, use a different strategy for the synthesis of AdoCbi-GDP (2). These organisms do not synthesize CobU; instead, they use CobY, a GTP:AdoCbi-P guanylyltransferase enzyme that lacks the NTP:AdoCbi-P kinase activity of CobU (Fig. 1) (14). The cobY gene was identified in extreme halophilic and thermophilic, methanogenic archaea as a non-orthologous replacement for cobU (14, 15). In this work we identified the locus encoding the CobY enzyme in the hyperthermophilic methanogenic archaeon Methanocaldococcus jannaschii. Recombinant protein was expressed in E. coli cells, the order of substrate binding was determined, and the interactions between substrate and enzyme were quantified.