The non-catalytic site Y700 regulates diguanylate cyclase activity of PA0847 in Pseudomonas aeruginosa

Abstract:

[Objective] To identify the diguanylate cyclase activity of the intracellular domain and its mutants of PA0847 from Pseudomonas aeruginosa and preliminarily probe into its catalytic mechanism. [Methods] Congo red plate staining was employed to verify the diguanylate cyclase activity of the intracellular domain of PA0847. PCR was employed to construct the PA0847 PAS-GGDEF domain and its single-point mutants, and the corresponding proteins were expressed and purified. Gel filtration chromatography was utilized to analyze the aggregation states of proteins in solution. The diguanylate cyclase activity of the proteins was identified by in-vitro enzymatic reactions. Based on thiazole orange fluorescence staining, the production of cyclic diguanylate monophosphate (c-di-GMP) was determined after the enzymatic reactions, and the amino acid residues closely related to the activity of diguanylate cyclase were screened. The structural models of PA0847 PAS-GGDEF and its complex with guanosine triphosphate (GTP) were obtained by structure prediction combined with molecular docking. [Results] PA0847 PAS-GGDEF primarily exerted its catalytic activity as a dimer, with the PAS domain facilitating the dimer formation and increasing the activity of the diguanylate cyclase. Mutant screening revealed a significant increase in the activity of the non-catalytic site mutant Y700A compared with the wild type at a low protein concentration (<0.6 mg/mL). Gel filtration chromatography indicated that the heightened activity may be attributed to the enhanced GGDEF (Gly-Gly-Asp-Glu-Phe) dimerization driven by Y700A. Structural modeling revealed that PA0847 PAS-GGDEF had a conserved GTP binding site, in which the amino side chain of K722 played an important role in binding to the phosphoryl group of GTP. The aromatic ring of Y700 engaged in a hydrophobic interaction with the alpha-helix containing K722. Therefore, the mutation Y700A may alter the spatial orientation of the K722-containing helix, which promoted the binding of K722 to the substrate GTP and the dimerization of GGDEF. [Conclusion] The non-catalytic site Y700 of PA0847 from Pseudomonas aeruginosa can indirectly regulate the diguanylate cyclase activity.