A non-canonical metal center drives activity of the Sediminispirochaeta smaragdinae metallo-β-lactamase SPS-1.

In an effort to evaluate whether a recently reported putative metallo-beta-lactamase (M beta L) contains a novel M beta L active site, SPS-1 from Sediminispirochaeta smaragdinae was overexpressed, purified, and characterized using spectroscopic and crystallographic studies. Metal analyses demonstrate that recombinant SPS-1 binds nearly 2 equiv of Zn(II), and steady-state kinetic studies show that the enzyme hydrolyzes carbapenems and certain cephalosporins but not beta-lactam substrates with bulky substituents at the 6/7 position. Spectroscopic studies of Co(II)-substituted SPS-1 suggest a novel metal center in SPS-1, with a reduced level of spin coupling between the metal ions and a novel Zn-I metal binding site. This site was confirmed with a crystal structure of the enzyme. The structure shows a Zn-2 site that is similar to that in NDM-1 and other subclass B1 M beta Ls; however, the Zn-1 metal ion is coordinated by two histidine residues and a water molecule, which is held in position by a hydrogen bond network. The Zn-1 metal is displaced nearly 1 angstrom from the position reported in other M beta Ls. The structure also shows extended helices above the active site, which create a binding pocket that precludes the binding of substrates with large, bulky substituents at the 6/7 position of beta-lactam antibiotics. This study reveals a novel metal binding site in M beta Ls and suggests that the targeting of metal binding sites in M beta Ls with inhibitors is now more challenging with the identification of this new M beta L.