Modification of a bi-functional diguanylate cyclase-phosphodiesterase to efficiently produce cyclic diguanylate monophosphate

Abstract

Cyclic-diGMP is a bacterial messenger that regulates many physiological processes, including many attributed to pathogenicity. Bacteria synthesize cyclic-diGMP from GTP using diguanylate cyclases; its hydrolysis is catalyzed by phosphodiesterases. Here we report the over-expression and purification of a bi-functional diguanylate cyclase-phosphodiesterase from Agrobacterium vitis S4. Using homology modeling and primary structure alignment, we identify several amino acids predicted to participate in the phosphodiesterase reaction. Upon altering selected residues, we obtain variants of the enzyme that efficiently and quantitatively catalyze the synthesis of cyclic-diGMP from GTP without hydrolysis to pGpG. Additionally, we identify a variant that produces cyclic-diGMP while immobilized to NiNTA beads and can catalyze the conversion of [α-³²P]-GTP to [³²P]-cyclic-diGMP. In short, we characterize a novel cyclic-diGMP processing enzyme and demonstrate its utility for efficient and cost-effective production of cyclic-diGMP, as well as modified cyclic-diGMP molecules, for use as probes in studying the many important biological processes mediated by cyclic-diGMP.

Keywords: AvHaCE, Agrobacterium vitis H-NOX associated cyclic-diGMP processing enzyme; Cyclic-diGMP; Cyclic-diGMP, bis-3′-5′-cyclic dimeric guanosine monophosphate; DGC, diguanylate cyclase; Diguanylate cyclase; GTP, guanosine-5′-triphosphate; H-NOX associated cyclic-diGMP processing enzyme; PDE, phosphodiesterase; Phosphodiesterase; [32P]-cyclic-diGMP; pGpG, 5′-phosphoguanylyl-(3′,5′)-guanosine).

Graphical abstract

Fig. 1

Characterization of wild-type AvHaCE. (A) Coomassie blue stained SDS-PAGE gel (12.5%) of purified AvHaCE. Lane 1, molecular mass standards; lane 2, AvHaCE after purification by immobilized metal affinity chromatography with Ni–NTA as the matrix. (B) HPLC analyses of wild-type AvHaCE reaction product and guanine nucleotide standards, as indicated. (C) MALDI analysis of the HPLC purified AvHaCE reaction product. The species with a molecular mass of 558 corresponds to pGp. (D) Time-course for pGpG production from GTP with wild-type AvHaCE. To correct for variations in injection volumes, pGpG and GTP peak areas were normalized to peak areas of a co-injected NAD standard.

Fig. 2

Residues important for phosphodiesterase activity. (A) HPLC analysis of the reaction products of the AVL variant of AvHaCE after incubation overnight. (B) HPLC analysis of the reaction products of the AAL variant of AvHaCE before and after incubation overnight. The HPLC analysis of the nucleotide mixture immediately after adding AvHaCE is also illustrated, indicting the retention time of GTP. (C) Primary structure alignment of AvHaCE and the phosphodiesterase YkuI from B. subtilis (BSU14090). Conserved residues are highlighted in red. Residues shown to be important for phosphodiesterase activity of RocR from P. aeruginosa are indicated by the asterisk. The yellow highlight indicates the position of loop D. (D) Homology model of residues 249-502, corresponding to the phosphodiesterase domain, of AvHaCE. The model was built with YkuI (pdb 2W27) as the template. Select amino acids proposed to be located within the phosphodiesterase active site of AvHaCE, as well as bound cyclic-diGMP, are rendered in stick format. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

HPLC analyses of reaction products of AvHaCE variant E461Q and D427A, as indicated. Reactions were overnight at room temperature as described in the text.

Fig. 4

Enzymatic synthesis of cyclic-diGMP with the AvHaCE AVL variant. (A) HPLC analysis of cyclic-diGMP standard. (B) HPLC analysis of reaction products at 0 min (dotted line), after 4 h incubation (dashed line), and overnight incubation (solid line) at room temperature. The species observed at 0 min is GTP.

Fig. 5

MALDI analysis of cyclic-diGMP synthesized by immobilized AvHaCE AAL variant.

Fig. 6

HPLC analysis of radiolabeled cyclic-diGMP synthesis by the AvHaCE AAL variant. Both radioactivity and UV–vis absorbance were monitored, as indicated. A slight shift in the detection of radioactivity and absorbance occurs because of the delay that occurs as eluted material flows first through one detector then the other. (A) Reaction products after 15 min incubation at room temperature. (B) Reaction products after 60 min incubation at room temperature.