title: Crystal structure of Pseudomonas aeruginosa SPM-1 provides insights into variable zinc affinity of metallo-beta-lactamases.
abstract: Metallo-beta-lactamases (mbetals) confer broad-spectrum resistance to beta-lactam antibiotics upon host bacteria and escape the action of existing beta-lactamase inhibitors. SPM-1 is a recently discovered mbetal that is distinguished from related enzymes by possession of a substantial central insertion and by sequence variation at positions that maintain active site structure. Biochemical data show SPM-1 to contain two Zn2+ sites of differing affinities, a phenomenon that is well documented amongst mbetals but for which a structural explanation has proved elusive. Here, we report the crystal structure of SPM-1 to 1.9 A resolution. The structure reveals SPM-1 to lack a mobile loop implicated in substrate binding by related mbetals and to accommodate the central insertion in an extended helical interdomain region. Deleting this had marginal effect upon binding and hydrolysis of a range of beta-lactams. These data suggest that the interactions of SPM-1 with substrates differ from those employed by other mbetals. SPM-1 as crystallised contains a single Zn2+. Both the active site hydrogen-bonding network and main-chain geometry at Asp120, a key component of the binding site for the second zinc ion, differ significantly from previous mbetal structures. We propose that variable interactions made by the Asp120 carbonyl group modulate affinity for a second Zn2+ equivalent in mbetals of the B1 subfamily. We further predict that SPM-1 possesses the capacity to evolve variants of enhanced catalytic activity by point mutations altering geometry and hydrogen bonding in the vicinity of the second Zn2+ site.
authors.affiliation: Department of Cellular and Molecular Medicine, University of Bristol School of Medical Sciences, University Walk, Bristol BS8 1TD, UK.
authors.forename: Tanya A
authors.initials: TA
authors.lastname: Murphy
authors.forename: Lucy E
authors.initials: LE
authors.lastname: Catto
authors.forename: Stephen E
authors.initials: SE
authors.lastname: Halford
authors.forename: Andrea T
authors.initials: AT
authors.lastname: Hadfield
authors.forename: Wladek
authors.initials: W
authors.lastname: Minor
authors.forename: Timothy R
authors.initials: TR
authors.lastname: Walsh
authors.forename: James
authors.initials: J
authors.lastname: Spencer
mesh_terms: D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D003460:Crystallization; D018360:Crystallography, X-Ray; D019281:Dimerization; D020836:Protein Structure, Quaternary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D015032:Zinc / Q000737:chemistry / Q000378:metabolism*; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: C476155:beta-lactamase SPM-1, Pseudomonas aeruginosa; D001618:beta-Lactamases; D015032:Zinc