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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.

Cobinamide (Cbi) salvaging is impaired, but not abolished, in a Salmonella enterica strain lacking a functional cobU gene. CobU is a bifunctional enzyme (NTP:adenosylcobinamide [NTP:AdoCbi] kinase, GTP:adenosylcobinamide-phosphate [GTP:AdoCbi-P] guanylyltransferase) whose AdoCbi kinase activity is necessary for Cbi salvaging in this bacterium. Inactivation of the ycfN gene in a Δ cobU strain abrogated Cbi salvaging. Introduction of a plasmid carrying the ycfN + allele into a Δ cobU Δ ycfN strain substantially restored Cbi salvaging. Mass spectrometry data indicate that when YcfN-enriched cell extracts were incubated with AdoCbi and ATP, the product of the reaction was AdoCbi-P. Results from bioassays confirmed that YcfN converted AdoCbi to AdoCbi-P in an ATP-dependent manner. YcfN is a good example of enzymes that are used by the cell in multiple pathways to ensure the salvaging of valuable precursors.

Bacterial spores are surrounded by a morphologically complex, mechanically flexible protein coat, which protects the spore from toxic molecules. The interactions among the over 50 proteins that make up the coat remain poorly understood. We have used cell biological and protein biochemical approaches to identify novel coat proteins in Bacillus subtilis and describe the network of their interactions, in order to understand coat assembly and the molecular basis of its protective functions and mechanical properties. Our analysis characterizes the interactions between 32 coat proteins. This detailed view reveals a complex interaction network. A key feature of the network is the importance of a small subset of proteins that direct the assembly of most of the coat. From an analysis of the network topology, we propose a model in which low-affinity interactions are abundant in the coat and account, to a significant degree, for the coat's mechanical properties as well as structural variation between spores.

GTP:adenosylcobinamide-phosphate (AdoCbi-P) guanylyl transferase (CobY) is an enzyme that transfers the GMP moiety of GTP to AdoCbi yielding AdoCbi-GDP in the late steps of the assembly of Ado-cobamides in archaea. The failure of repeated attempts to crystallize ligand-free (apo) CobY prompted us to explore its 3D structure by solution NMR spectroscopy. As reported here, the solution structure has a mixed α/β fold consisting of seven β-strands and five α-helices, which is very similar to a Rossmann fold. Titration of apo-CobY with GTP resulted in large changes in amide proton chemical shifts that indicated major structural perturbations upon complex formation. However, the CobY:GTP complex as followed by 1H-15N HSQC spectra was found to be unstable over time: GTP hydrolyzed and the protein converted slowly to a species with an NMR spectrum similar to that of apo-CobY. The variant CobYG153D, whose GTP complex was studied by X-ray crystallography, yielded NMR spectra similar to those of wild-type CobY in both its apo- state and in complex with GTP. The CobYG153D:GTP complex was also found to be unstable over time.