Abstract
The bacterial second messenger cyclic di-3′,5′-guanosine monophosphate (c-di-GMP) is a key regulator of bacterial motility and virulence. As high levels of c-di-GMP are associated with the biofilm lifestyle, c-di-GMP hydrolysing phosphodiesterases (PDEs) have been identified as key targets to aid development of novel strategies to treat chronic infection by exploiting biofilm dispersal. We have studied the EAL signature motif-containing phosphodiesterase domains from the Pseudomonas aeruginosa proteins PA3825 (PA3825EAL) and PA1727 (MucREAL). Different dimerisation interfaces allow us to identify interface independent principles of enzyme regulation. Unlike previously characterised two-metal binding EAL-phosphodiesterases, PA3825EAL in complex with pGpG provides a model for a third metal site. The third metal is positioned to stabilise the negative charge of the 5′-phosphate, and thus three metals could be required for catalysis in analogy to other nucleases. This newly uncovered variation in metal coordination may provide a further level of bacterial PDE regulation.
Highlights
Cyclic di-3′,5′-guanosine monophosphate (c-di-GMP) is a universal second messenger in eubacteria that regulates a wide variety of functions including formation and dispersal of biofilms[1,2,3,4], adhesion[1,5,6], motility[7,8,9], synthesis of virulence factors[10] and developmental transitions[11,12]
Hydrolysis of c-di-GMP by EAL domains has been shown to require the presence of divalent ions, either Mg2+ or Mn2+, with the most efficient reactions occurring at alkaline pH29,33
Dimerisation of the EAL domain YahA from Escherichia Coli has been shown to increase substrate-turnover, while specific site directed mutagenesis preventing dimerisation results in reduced PDE activity; without altering the substrate binding affinity[33]. This regulation is further rationalised through observations of EAL domain dimerisation in P. aeruginosa MorA, which causes the α5 helix to unwind at the dimerisation interface; allowing the catalytic DDFGTG motif on the β5-α5 loop to enter the substrate binding pocket and coordinate catalytically important metal ions in the active site[35]
Summary
Cyclic di-3′,5′-guanosine monophosphate (c-di-GMP) is a universal second messenger in eubacteria that regulates a wide variety of functions including formation and dispersal of biofilms[1,2,3,4], adhesion[1,5,6], motility[7,8,9], synthesis of virulence factors[10] and developmental transitions[11,12]. Dimerisation of the EAL domain YahA from Escherichia Coli has been shown to increase substrate-turnover, while specific site directed mutagenesis preventing dimerisation results in reduced PDE activity; without altering the substrate binding affinity[33] This regulation is further rationalised through observations of EAL domain dimerisation in P. aeruginosa MorA, which causes the α5 helix to unwind at the dimerisation interface; allowing the catalytic DDFGTG motif on the β5-α5 loop (previously labelled as connecting β6 and α627,34) to enter the substrate binding pocket and coordinate catalytically important metal ions in the active site[35]. Crystal structures of dimers have predominantly been reported with bound substrate, c-di-GMP28,29,37 This is not consistent with a model of activation through dimer formation and suggests that further regulatory steps must be required for the activation of EAL-PDEs. A model organism for study of biofilm dispersal, and EAL-PDE behaviour, is Pseudomonas aeruginosa PAO1. We characterise a potential third metal binding site that directly interacts with the reaction product pGpG
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
