title: Dimerisation induced formation of the active site and the identification of three metal sites in EAL-phosphodiesterases.
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, PA3825EALin 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.
authors.affiliation: Diamond Light Source, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0FA, United Kingdom.|Research Complex at Harwell, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0FA, United Kingdom.
authors.forename: Dom
authors.initials: D
authors.lastname: Bellini
authors.affiliation: Diamond Light Source, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0FA, United Kingdom.|School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, CO4 3SQ, Essex, United Kingdom.
authors.forename: Sam
authors.initials: S
authors.lastname: Horrell
authors.affiliation: Diamond Light Source, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0FA, United Kingdom.|Centre for Biological Sciences and Institute for Life Sciences, Life Sciences Building B85, The University of Southampton, University Rd, Southampton, Hampshire, SO17 1BJ, United Kingdom.
authors.forename: Andrew
authors.initials: A
authors.lastname: Hutchin
authors.affiliation: Centre for Biological Sciences and Institute for Life Sciences, Life Sciences Building B85, The University of Southampton, University Rd, Southampton, Hampshire, SO17 1BJ, United Kingdom.
authors.forename: Curtis W
authors.initials: CW
authors.lastname: Phippen
authors.affiliation: School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, CO4 3SQ, Essex, United Kingdom.
authors.forename: Richard W
authors.initials: RW
authors.lastname: Strange
authors.affiliation: Centre for Biological Sciences and Institute for Life Sciences, Life Sciences Building B85, The University of Southampton, University Rd, Southampton, Hampshire, SO17 1BJ, United Kingdom.
authors.forename: Yuming
authors.initials: Y
authors.lastname: Cai
authors.affiliation: Diamond Light Source, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0FA, United Kingdom.
authors.forename: Armin
authors.initials: A
authors.lastname: Wagner
authors.affiliation: Centre for Biological Sciences and Institute for Life Sciences, Life Sciences Building B85, The University of Southampton, University Rd, Southampton, Hampshire, SO17 1BJ, United Kingdom.
authors.forename: Jeremy S
authors.initials: JS
authors.lastname: Webb
authors.affiliation: Centre for Biological Sciences and Institute for Life Sciences, Life Sciences Building B85, The University of Southampton, University Rd, Southampton, Hampshire, SO17 1BJ, United Kingdom.
authors.forename: Ivo
authors.initials: I
authors.lastname: Tews
authors.affiliation: Diamond Light Source, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0FA, United Kingdom.|Research Complex at Harwell, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0FA, United Kingdom.
authors.forename: Martin A
authors.initials: MA
authors.lastname: Walsh
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D020134:Catalytic Domain; D002413:Cations, Divalent; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry* / Q000378:metabolism; D008345:Manganese / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D011550:Pseudomonas aeruginosa / Q000737:chemistry* / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D002413:Cations, Divalent; D011994:Recombinant Proteins; D008345:Manganese; C062025:bis(3',5')-cyclic diguanylic acid; D010727:Phosphoric Diester Hydrolases; D006152:Cyclic GMP; D008274:Magnesium