title: Structural and functional studies of a metallo-β-lactamase unveil a new type of structurally encoded nickel-containing heterodinuclear site.
abstract: The selection of correct metal ions with high fidelity against competing cellular cations is crucial for the function of many metalloenzymes; however, the understanding of the principles that govern metal selectivity is still incomplete. In this study, the crystal structure of the Tm1162 protein from Thermotoga maritima, a metallo-β-lactamase, is reported. Several crystal structures of wild-type Tm1162 and its mutants were solved. Homologues of Tm1162 are widely distributed in bacteria and archaea, including several human pathogens. The monomer possesses an αβ/βα fold, with the core β-strands having the β-sheet sandwich structure common to the metallo-β-lactamase superfamily. Tm1162 exists as a trimer in the crystal and this trimeric unit is likely to be present in solution. In the trimer, three active sites reside at the interface between subunits, suggesting that the oligomeric assembly is crucial for catalysis. A new type of structurally encoded heterodinuclear site has been identified by confirming the identity of nickel-containing heteronuclear sites in Tm1162 via X-ray absorption spectroscopy and anomalous difference Fourier maps. The second coordination sphere, including His8 and Glu73, maintains the side-chain orientations of histidines and stabilizes the metal-binding site. Nickel coordination was crucial for the oligomerization of Tm1162. The nickel-dependent and manganese-dependent β-lactamase and phosphodiesterase activities of Tm1162 have also been characterized.
authors.affiliation: Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-742, Republic of Korea.
authors.forename: Hwajung
authors.initials: H
authors.lastname: Choi
authors.affiliation: Department of Bio and Nano Chemistry, Kookmin University, Seoul 136-702, Republic of Korea.
authors.forename: Hee Jung
authors.initials: HJ
authors.lastname: Kim
authors.affiliation: Department of Bio and Nano Chemistry, Kookmin University, Seoul 136-702, Republic of Korea.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Matsuura
authors.affiliation: Laboratory of Quality Design and Exploitation, Division of Agronomy and Horticultural Science, Graduate School of Agriculture, Kyoto University, Kyoto 611-0011, Japan.
authors.forename: Bunzo
authors.initials: B
authors.lastname: Mikami
authors.affiliation: Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-742, Republic of Korea.
authors.forename: Hye Jin
authors.initials: HJ
authors.lastname: Yoon
authors.affiliation: Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-742, Republic of Korea.
authors.forename: Hyung Ho
authors.initials: HH
authors.lastname: Lee
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009532:Nickel / Q000737:chemistry* / Q000378:metabolism; D016415:Sequence Alignment; D020124:Thermotoga maritima / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D056928:X-Ray Absorption Spectroscopy; D001618:beta-Lactamases / Q000737:chemistry* / Q000378:metabolism
chemical_list: D009532:Nickel; D001618:beta-Lactamases
keywords: X-ray absorption spectroscopy; antibiotics; metalloenzyme; nickel; phosphodiesterase