title: Ca(II) Binding Regulates and Dominates the Reactivity of a Transition-Metal-Ion-Dependent Diesterase from Mycobacterium tuberculosis.
abstract: The diesterase Rv0805 from Mycobacterium tuberculosis is a dinuclear metallohydrolase that plays an important role in signal transduction by controlling the intracellular levels of cyclic nucleotides. As Rv0805 is essential for mycobacterial growth it is a promising new target for the development of chemotherapeutics to treat tuberculosis. The in vivo metal-ion composition of Rv0805 is subject to debate. Here, we demonstrate that the active site accommodates two divalent transition metal ions with binding affinities ranging from approximately 50 nm for Mn(II) to about 600 nm for Zn(II) . In contrast, the enzyme GpdQ from Enterobacter aerogenes, despite having a coordination sphere identical to that of Rv0805, binds only one metal ion in the absence of substrate, thus demonstrating the significance of the outer sphere to modulate metal-ion binding and enzymatic reactivity. Ca(II) also binds tightly to Rv0805 (Kd ≈40 nm), but kinetic, calorimetric, and spectroscopic data indicate that two Ca(II) ions bind at a site different from the dinuclear transition-metal-ion binding site. Ca(II) acts as an activator of the enzymatic activity but is able to promote the hydrolysis of substrates even in the absence of transition-metal ions, thus providing an effective strategy for the regulation of the enzymatic activity.
authors.affiliation: School of Chemistry and Molecular BioSciences, The University of Queensland, St. Lucia, QLD 4072, Australia), Fax.
authors.forename: Marcelo M
authors.initials: MM
authors.lastname: Pedroso
authors.affiliation: Department of Chemistry and Biochemistry, Middlebury College, Middlebury, VT 05753, USA.
authors.forename: James A
authors.initials: JA
authors.lastname: Larrabee
authors.affiliation: School of Chemistry and Molecular BioSciences, The University of Queensland, St. Lucia, QLD 4072, Australia), Fax.
authors.forename: Fernanda
authors.initials: F
authors.lastname: Ely
authors.affiliation: School of Chemistry and Molecular BioSciences, The University of Queensland, St. Lucia, QLD 4072, Australia), Fax.
authors.forename: Shuhui E
authors.initials: SE
authors.lastname: Gwee
authors.affiliation: Department of Chemistry, National University of Ireland-Maynooth, Maynooth, Co. Kildare, Ireland.
authors.forename: Nataša
authors.initials: N
authors.lastname: Mitić
authors.affiliation: Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia.
authors.forename: David L
authors.initials: DL
authors.lastname: Ollis
authors.affiliation: School of Chemistry and Molecular BioSciences, The University of Queensland, St. Lucia, QLD 4072, Australia), Fax.
authors.forename: Lawrence R
authors.initials: LR
authors.lastname: Gahan
authors.affiliation: School of Chemistry and Molecular BioSciences, The University of Queensland, St. Lucia, QLD 4072, Australia), Fax. schenk@uq.edu.au.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Schenk
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry*; D007477:Ions / Q000737:chemistry*; D009169:Mycobacterium tuberculosis / Q000737:chemistry* / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D028561:Transition Elements / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D007477:Ions; C000608471:Rv0805 protein, Mycobacterium tuberculosis; D028561:Transition Elements; D010727:Phosphoric Diester Hydrolases; C038133:glycerophosphodiester phosphodiesterase; D002118:Calcium
keywords: calcium; coordination spheres; dinuclear metallohydrolases; enzyme catalysis; organophosphate pesticides