Use of ferrous iron by metallo-β-lactamases

Samuel T Cahill,Hanna Tarhonskaya,Anna M Rydzik,Emily Flashman,Michael A Mcdonough,Christopher J Schofield,Jürgen Brem

doi:10.1016/j.jinorgbio.2016.07.013

Abstract

Metallo-β-lactamases (MBLs) catalyse the hydrolysis of almost all β-lactam antibacterials including the latest generation carbapenems and are a growing worldwide clinical problem. It is proposed that MBLs employ one or two zinc ion cofactors in vivo. Isolated MBLs are reported to use transition metal ions other than zinc, including copper, cadmium and manganese, with iron ions being a notable exception. We report kinetic and biophysical studies with the di-iron(II)-substituted metallo-β-lactamase II from Bacillus cereus (di-Fe(II) BcII) and the clinically relevant B1 subclass Verona integron-encoded metallo-β-lactamase 2 (di-Fe(II) VIM-2). The results reveal that MBLs can employ ferrous iron in catalysis, but with altered kinetic and inhibition profiles compared to the zinc enzymes. A crystal structure of di-Fe(II) BcII reveals only small overall changes in the active site compared to the di-Zn(II) enzyme including retention of the di-metal bridging water; however, the positions of the metal ions are altered in the di-Fe(II) compared to the di-Zn(II) structure. Stopped-flow analyses reveal that the mechanism of nitrocefin hydrolysis by both di-Fe(II) BcII and di-Fe(II) VIM-2 is altered compared to the di-Zn(II) enzymes. Notably, given that the MBLs are the subject of current medicinal chemistry efforts, the results raise the possibility the Fe(II)-substituted MBLs may be of clinical relevance under conditions of low zinc availability, and reveal potential variation in inhibitor activity against the differently metallated MBLs.

Highlights

More than 70 years after their first clinical application, the β-lactams remain the most important antibacterials in use [1]. β-Lactamases constitute an important mode of resistance to β-lactam antibacterials by catalysing hydrolysis of the β-lactam ring to give inactive β-amino acid products [2,3,4,5]
The β-lactamase activity of di-Fe(II) metallo-βlactamase II from Bacillus cereus (BcII) is comparable to that of the di-Zn(II) enzyme, in terms of kcat/Km values, with both substrates. In these assays Verona integron-encoded metallo-β-lactamase 2 (VIM-2) shows around a 10-fold lower efficiency with both substrates when using the di-Fe(II) enzyme compared to the di-Zn(II) enzyme
The results reveal that the class B1 MBLs are able to bind Fe(II) and, in contrast to previous reports [11,22,23,24], are active when their Zn(II) ions are replaced by Fe(II) ions under low oxygen conditions, with the enzymes being able to hydrolyse both the chromogenic substrate nitrocefin and the clinically employed carbapenem antibiotic meropenem

Summary

Introduction

More than 70 years after their first clinical application, the β-lactams remain the most important antibacterials in use [1]. β-Lactamases constitute an important mode of resistance to β-lactam antibacterials by catalysing hydrolysis of the β-lactam ring to give inactive β-amino acid products [2,3,4,5]. Β-lactamases are divided into two classes, i.e. those that employ a nucleophilic serine residue (serine β-lactamases (SBLs), Ambler classes A, C, and D) and those requiring metal ions for hydrolysis (metallo-β-lactamases (MBLs), Ambler class B) [6,7]. The true MBLs, i.e. those catalysing β-lactam hydrolysis, are further divided into three subclasses, B1, B2, and B3. Both B1 and B3 enzymes bind two Zn(II) ions in their native state, with the exception of the B3 enzyme GOB, which can exhibit activity when a single Zn(II) ion is bound [11], whereas B2 enzymes bind one Zn(II) ion and are inhibited through binding of a second ion [12,13]. A water molecule, proposed to be a hydroxide ion, Wat, bridges the two metal centres while an additional ‘terminal’ water molecule, Wat, is bound to Zn2 (Fig. 1) [15]

Methods

Results

Conclusion