A bioinformatic analysis of the GGDEF domain sequence and structure published in 2001 by Pei and Grishin (109) was also useful selleck screening library in connecting this domain to the cyclase activity. These authors discovered that the GGDEF domain is distantly related to the catalytic domain of adenylate/guanylate nucleotide cyclases (110, 111). While primary sequence similarity between these domains is low, the predicted secondary and tertiary structures of the GGDEF domain are remarkably similar to those of the type III adenylate cyclase. Pei and Grishin proposed that the GGDEF domain is a DGC and predicted the loop involving the most conserved signature motif, GG(D/E)EF, to be part of the substrate (GTP) binding site. The first biochemical evidence solidifying this connection came from a study by Paul et al.

(37), who showed that the phosphorylated form of PleD converts GTP into c-di-GMP in vitro. This was also observed by Hickman et al. (93) and Ryjenkov et al. (42). The latter study analyzed in vitro activities of six different GGDEF domain enzymes originating from representatives of diverse branches of the bacterial phylogenetic tree, including Alpha- and Gammaproteobacteria as well as Thermotogae, Deinococcus-Thermus, Cyanobacteria, and Spirochaetes. All of these GGDEF domain proteins possessed DGC activity and were incapable of utilizing nucleotide substrates other than GTP. Therefore, the ubiquity and evolutionary conservation of c-di-GMP envisioned earlier (25) were established experimentally (Fig. 2). Fig 2 Basic biochemistry of c-di-GMP synthesis, degradation, and c-di-GMP receptors.

The diagrams show the protein domains involved in c-di-GMP metabolism and signaling. Enzymatically active GGDEF, EAL, and HD-GYP domains are shown on a white background. Enzymatically … How do GGDEF domain proteins catalyze c-di-GMP formation? The early insights into this question were obtained by Benziman and colleagues (1), who revealed that c-di-GMP formation from 2 molecules of GTP is a two-step reaction proceeding via 5��-pppGpG as a reaction intermediate (Fig. 2). Two molecules of pyrophosphate are reaction by-products. A further mechanistic understanding of c-di-GMP synthesis came from the biochemical and structural characterization of DGCs. The apparent similarity of DGCs to type III nucleotide cyclases, as well as the dinucleotide nature of c-di-GMP, implied that GGDEF domains function as homodimers, where two monomers come together to form an active site at the dimer interface (112). Each GGDEF monomer contributes a GTP substrate to the formation of an intermolecular Carfilzomib phosphoester bond to another molecule of GTP. It was observed that purified GGDEF domains by themselves form homodimers and, at high concentrations, show low-level DGC activity.