Mutagenesis of Zinc Ligand Residue Cys221 Reveals Plasticity in the IMP-1 Metallo-β-Lactamase Active Site

Abstract

e Metallo-␤-lactamases catalyze the hydrolysis of a broad range of ␤-lactam antibiotics and are a concern for the spread of drug resistance. To analyze the determinants of enzyme structure and function, the sequence requirements for the subclass B1 IMP-1 ␤-lactamase zinc binding residue Cys221 were tested by saturation mutagenesis and evaluated for protein expression, as well as hydrolysis of ␤-lactam substrates. The results indicated that most substitutions at position 221 destabilized the enzyme. Only the enzymes containing C221D and C221G substitutions were expressed well in Escherichia coli and exhibited catalytic activity toward ␤-lactam antibiotics. Despite the lack of a metal-chelating group at position 221, the C221G enzyme exhibited high levels of catalytic activity in the presence of exogenous zinc. Molecular modeling suggests the glycine substitution is unique among substitutions in that the complete removal of the cysteine side chain allows space for a water molecule to replace the thiol and coordinate zinc at the Zn2 zinc binding site to restore function. Multiple methods were used to estimate the C221G Zn2 binding constant to be 17 to 43 M. Studies of enzyme function in vivo in E. coli grown on minimal medium showed that both IMP-1 and the C221G mutant exhibited compromised activity when zinc availability was low. Finally, substitutions at residue 121, which is the IMP-1 equivalent of the subclass B3 zinc-chelating position, failed to rescue C221G function, suggesting the coordination schemes of subclasses B1 and B3 are not interchangeable.A n increasing prevalence of antibiotic-resistant strains is reducing the available options for treating bacterial infections. ␤-Lactam antibiotics, such as the penicillins and cephalosporins, are among the most often used antimicrobial agents (31). The major contributors to ␤-lactam antibiotic resistance are ␤-lactamase enzymes, which act by hydrolyzing the four-member ␤-lactam ring (15,62). Mechanistically, this is accomplished either via an active-site serine in the class A, C, and D enzymes or through the use of one or two Zn 2ϩ ions (class B) (4). Class B metallo-␤-lactamases (M␤Ls) are a group of structurally similar enzymes that exhibit a characteristic ␣␤/␤␣ sandwich fold, with the active site located at the interface between domains. This scaffold supports up to 6 residues at the active site that coordinate either one or two zinc ions that are central to the catalytic mechanism (1,6,35). M␤Ls have the capacity to hydrolyze most clinically available ␤-lactam drugs, including extended-spectrum cephalosporins and carbapenems (8,10,32,40,59,61).The IMP-1 metallo-␤-lactamase has been identified in several nosocomial, Gram-negative, pathogenic bacteria, including Pseudomonas aeruginosa and Serratia marcesens (22, 28). The bla IMP-1 gene is carried on an integron element that facilitates genetic transfer and is likely why IMP-1 is found among multiple bacterial species (28, 58). The combination of a broad ␤-lactam antibiotic substrate profile and the genetic ca...

Metallo-β-Lactamases: A Major Threat to Human Health

“…As mentioned above, Badarau and Page reported K d values for Zn1 and Zn2 in BcII [111], and in a more recent report Horton et al employed ITC to measure K d2 (i.e. the K d of the Zn2 site) of the C221G mutant of IMP-1, which was purified with the Zn1 site occupied by a zinc ion [48]. The binding constant was estimated to be 17 μM, indicating that the cysteine ligand may not play a major role in binding the metal ion in Zn2 in B1-type MBLs, an observation in agreement with the proposed positive cooperativity for metal ion binding in the subgroup.…”

Understanding the determinants of substrate specificity in IMP family metallo‐β‐lactamases: The importance of residue 262

“…Other sequence-structure-activity studies of the IMP-type b-lactamases have focused mostly on determining residues critical for activity, [22][23][24] responsible for an altered substrate profile, 25 or important for the hydrolysis of specific antibiotics. 26 Interestingly, in the IMP-1 b-lactamase two residues that tolerate mutations, even though they are highly conserved, have been heretofore identified: N233, 27 which has previously been assigned a role in forming an oxyanion hole, 21,28 and C221, 29 which is a functionally important Zn(II) ligand. 21,22 Attempts have been made to predict enzyme activity of IMP variants using molecular dynamics simulations 13 and computational protein design.…”

An altered zinc-binding site confers resistance to a covalent inactivator of New Delhi metallo-beta-lactamase-1 (NDM-1) discovered by high-throughput screening

“…12 This residue is a conserved primary zinc ligand in the metallo-beta-lactamase B1 family (Figure 5), 20 so we chose variants that might also serve as metal ion ligands. Substitution of Cys with Ser or His was deleterious both to activity and to zinc content (Table 2).…”

“…11 However, a recent study with another B1 family member (IMP-1) indicates that, despite its sequence conservation, the Cys residue at this site can be substituted by Asp or Gly and still confer resistance to β-lactams. 20 If variation of a single residue can also confer resistance against Cys-directed inhibitors, it may suggest that this is not a suitable strategy for therapeutic development. Therefore, we built on our HTS results to test this possibility with NDM-1 by using p-CMB ( 1 ) as a model inhibitor that covalently targets Cys residues.…”