Cyclic di-GMP (c-di-GMP) is a ubiquitous bacterial second messenger that is involved in the regulation of cell surface-associated traits and the persistence of infections. Omnipresent GGDEF and EAL domains, which occur in various combinations with regulatory domains, catalyze c-di-GMP synthesis and degradation, respectively. The crystal structure of full-length YkuI from Bacillus subtilis, composed of an EAL domain and a C-terminal PAS-like domain, has been determined in its native form and in complex with c-di-GMP and Ca 2؉. The EAL domain exhibits a triose-phosphate isomerase-barrel fold with one antiparallel ␤-strand. The complex with c-di-GMP-Ca 2؉ defines the active site of the putative phosphodiesterase located at the C-terminal end of the ␤-barrel. The EAL motif is part of the active site with Glu-33 of the motif being involved in cation coordination. The structure of the complex allows the proposal of a phosphodiesterase mechanism, in which the divalent cation and the general base Glu-209 activate a catalytic water molecule for nucleophilic in-line attack on the phosphorus. The C-terminal domain closely resembles the PASfold. Its pocket-like structure could accommodate a yet unknown ligand. YkuI forms a tight dimer via EAL-EAL and trans EAL-PASlike domain association. The possible regulatory significance of the EAL-EAL interface and a mechanism for signal transduction between sensory and catalytic domains of c-di-GMP-specific phosphodiesterases are discussed.

Background:The bacterial second messenger cyclic di-GMP (c-di-GMP) is degraded by EAL phosphodiesterases. Results:The isolated EAL domain is active only as a homodimer. Substrate binding is coupled with EAL dimerization. Conclusion: Activity of many full-length EAL phosphodiesterases may be regulated by catalytic domain dimerization. Significance: A generic mechanism for the regulation of a central node of c-di-GMP signaling is provided.

Cyclic di-GMP (c-di-GMP) is an almost universal bacterial second messenger involved in the regulation of cell surface-associated traits and the persistence of infections. GGDEF and EAL domain-containing proteins catalyse c-di-GMP synthesis and degradation, respectively. We report the enzymatic large-scale synthesis of c-di-GMP, making use of the GGDEF domain-containing protein YdeH from Escherichia coli. Overexpression and purification of YdeH have been established, and the conditions for c-di-GMP synthesis were optimised. In contrast to the chemical synthesis of c-di-GMP, enzymatic c-di-GMP production is a one-step reaction that can easily be performed with the equipment of a standard biochemical lab. The protocol allows the production of milligram amounts of c-di-GMP within 1 day and paves the way for extensive biochemical and biophysical studies on c-di-GMP-mediated processes.

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