X-ray Absorption Spectroscopy of the Zinc-Binding Sites in the Class B2 Metallo-β-lactamase ImiS from Aeromonas veronii bv. sobria

Abstract

X-ray absorption spectroscopy was used to investigate the metal-binding sites of ImiS from Aeromonas Veronii bV. sobria in catalytically active (1-Zn), product-inhibited (1-Zn plus imipenem), and inactive (2-Zn) forms. The first equivalent of zinc(II) was found to bind to the consensus Zn 2 site. The reaction of 1-Zn ImiS with imipenem leads to a product-bound species, coordinated to Zn via a carboxylate group. The inhibitory binding site of ImiS was examined by a comparison of wild-type ImiS with 1 and 2 equiv of bound zinc. 2-Zn ImiS extended X-ray absorption fine structure data support a binding site that is distant from the active site and contains both one sulfur donor and one histidine ligand. On the basis of the amino acid sequence of ImiS and the crystal structure of CphA [Garau et al. (2005) J. Mol. Biol. 345,[785][786][787][788][789][790][791][792][793][794][795], we propose that the inhibitory binding site is formed by M146, found on the B2-distinct R3 helix, and H118, a canonical Zn 1 ligand, proposed to help activate the nucleophilic water. The mutation of M146 to isoleucine abolishes metal inhibition. This is the first characterization of ImiS with the native metal Zn and establishes, for the first time, the location of the inhibitory metal site.

Carbapenemases: the Versatile β-Lactamases

“…Amino acid sequence identities are as low as 23% across the metallo-␤-lactamases, including enzymes such as CcrA, CphA, and L1 (Table 1) (182,224). However, all of the enzymes have conserved residues that bind zinc at two sites (see "Classification Schemes," above, and Table 1) and exhibit a well-conserved active site, as demonstrated through sophisticated modeling analyses (127) and in the crystal structures published to date (31,32,34,44,52,141,214).…”

Updated Functional Classification of β-Lactamases

“…Mechanistically, these enzymes are most effective in hydrolyzing carbapenems if only one of the zinc binding sites is occupied (26). In contrast to the other subgroups of MBLs, the presence of a second zinc ion is actually inhibitory to enzymatic activity (20).…”

Carbapenems: Past, Present, and Future

“…The CphA ␤-lactamase is a strict carbapenemase of subclass B2. CphA has been crystallized with one Zn 2ϩ ion, two Zn 2ϩ ions, and a biapenem intermediate trapped in the active site (15,16,40,67,208,209,(250)(251)(252). Previously, the second inhibitory Zn 2ϩ binding site was postulated to be remote from the active site (40,42).…”