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-β-lactamase (MβL) contains a novel MβL active site, SPS-1 from Sediminispirochaeta smaragdinae was over-expressed, purified, and characterized using spectroscopic and crystallographic studies. Metal analyses demonstrate that recombinant SPS-1 binds nearly 2 equivalents of Zn(II), and steady-state kinetic studies show that the enzyme hydrolyzes carbapenems and certain cephalosporins but not β-lactam substrates with bulky substituents in the 6/7 position. Spectroscopic studies on Co(II)-substituted SPS-1 suggest a novel metal center in SPS-1, with reduced spin coupling between the metal ions and a novel Zn 1 metal binding site. This site was confirmed with a crystal structure of the enzyme. The structure shows a Zn 2 site that is similar that that in NDM-1 and other subclass B1 MβLs; however, the Zn 1 metal ion is coordinated by 2 histidine residues and a water molecule, which is held in position by a hydrogen bond network. The Zn 1 metal is displaced nearly 1 Å from the position reported in other MβLs. 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 in the 6/7 position of β-lactam antibiotics. This study reveals a novel metal binding site in MβLs, and suggests that the targeting of metal binding sites in MβLs with inhibitors is now more challenging with the identification of this new MβL.