Enzymatic synthesis of cyclic dinucleotide analogs by a promiscuous cyclic-AMP-GMP synthetase and analysis of cyclic dinucleotide responsive riboswitches

Abstract

Cyclic dinucleotides are second messenger molecules produced by both prokaryotes and eukaryotes in response to external stimuli. In bacteria, these molecules bind to RNA riboswitches and several protein receptors ultimately leading to phenotypic changes such as biofilm formation, ion transport and secretion of virulence factors. Some cyclic dinucleotide analogs bind differentially to biological receptors and can therefore be used to better understand cyclic dinucleotide mechanisms in vitro and in vivo. However, production of some of these analogs involves lengthy, multistep syntheses. Here, we describe a new, simple method for enzymatic synthesis of several 3', 5' linked cyclic dinucleotide analogs of c-di-GMP, c-di-AMP and c-AMP-GMP using the cyclic-AMP-GMP synthetase, DncV. The enzymatic reaction efficiently produced most cyclic dinucleotide analogs, such as 2'-amino sugar substitutions and phosphorothioate backbone modifications, for all three types of cyclic dinucleotides without the use of protecting groups or organic solvents. We used these novel analogs to explore differences in phosphate backbone and 2'-hydroxyl recognition between GEMM-I and GEMM-Ib riboswitches.

Figure 1.

Mechanism of DncV catalysis. (A) Chemical structure of three known natural cyclic dinucleotides. The adenine base is indicated by green and the guanine base is shaded purple. (B) Cartoon representation of base recognition and chemical mechanism of c-AMP-GMP 3′-5′-phosphodiester bond based on previous crystal structures (23,24,26). Residue, D193, of the catalytic triad is highlighted in red. Hydrogen bonds are indicated by dashed lines. Interactions between the triphosphate backbone of the ligand and divalent ion are represented by dashed, orange lines.

Figure 2.

Base substitutions to cyclic dinucleotides and divalent ion optimization. (A) Base substitutions included on nucleotide triphosphates. (B) Divalent ion screen of DncV using GTP as substrate. (C) Cyclization of base analog nucleotide triphosphates using different divalent ions.

Figure 3.

Enzymatic synthesis of phosphorothioate c-di-AMP analogs. (A) Chemical structure of c-di-AMP phosphorothioate analogs. Conformation of the thiol groups is shown in red. (B) Chromatogram of chemically synthesized c-di-AMP phosphorothioates. (C) Chromatogram of enzymatically synthesized c-(R_pS_p)-A_ps in the presence of MnCl₂ from α-R_p-ATP and α-S_p-ATP. (D) Chromatograms of enzymatically synthesized c-(R_pR_p)-A_ps from α-S_p-ATP in the presence of MnCl₂ (blue trace) and CoCl₂ (red trace). (E) Chromatograms of enzymatically synthesized c-(S_pS_p)-A_ps from α-R_p-ATP in the presence of MnCl₂ (blue trace) and CoCl₂ (red trace).

Figure 4.

2′-hydroxyl and phosphate backbone recognition of c-di-GMP and c-AMP-GMP in GEMM-I and GEMM-Ib riboswitches. (A) Hydrogen bonding to 2′-hydroxyl and phosphate backbone of c-di-GMP in the GEMM-I riboswitch, VC2, from pdb file 3muh (44). The riboswitch is pictured in gray and cyclic-di-GMP is shown in purple. (B) Hydrogen bonding to 2′-hydroxyl and phosphate backbone in GEMM-Ib riboswitch, Gs1761, from pdb file 4yaz (33). The riboswitch is pictured in gray and cyclic-AMP-GMP is shown in cyan.