Controlling phosphodiesterase activity in biofilm dispersal (360G-Wellcome-207277_Z_17_Z)

Biofilm formation and dispersal are controlled by the secondary messenger bis-(3’-5’) cyclic dimeric guanosine monophosphate (c-di-GMP); high levels are associated with biofilm formation, while a reduction induces dispersal. The enzymes catalysing formation of c-di-GMP are diguanylate cyclases, while phosphodiesterases catalyse the breakdown of c-di-GMP. Structural studies of the EAL type phosphodiesterases were observed in the host laboratory in the presence of substrate c-di-GMP, suggesting that these structures would require further steps to attain full catalytic activity. The host laboratory have recently demonstrated that dimerization and active site formation as well as formation of three metal binding sites are distinct activation steps required to tune the enzymatic activity of phosphodiesterases (Scientific Reports 2017). The proposed project deals with the motility regulator protein, MorA, and the mucoid alginate regulator, MucR, from Pseudomonas aeruginosa. Both proteins have dual activity as diguanylate cyclase and phosphodiesterase. Interestingly, the two linked catalytic domains influence each other's activity; different levels of activity are observed for the phosphodiesterases when comparing isolated domains with the linked conformation. The objective of the project is to purify these proteins and determine their catalytic activities, comparing the isolated domains, mixtures of the individual domains, and the cyclase-phosphodiesterase domains in linked conformation.