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- Materials and methods
Metallo-β-lactamases have raised concerns due to their ability to hydrolyze a broad spectrum of β-lactam antibiotics. The G262S point mutation distinguishing the metallo-β-lactamase IMP-1 from IMP-6 has no effect on the hydrolysis of the drugs cephalothin and cefotaxime, but significantly improves catalytic efficiency toward cephaloridine, ceftazidime, benzylpenicillin, ampicillin, and imipenem. This change in specificity occurs even though residue 262 is remote from the active site. We investigated the substrate specificities of five other point mutants resulting from single-nucleotide substitutions at positions near residue 262: G262A, G262V, S121G, F218Y, and F218I. The results suggest two types of substrates: type I (nitrocefin, cephalothin, and cefotaxime), which are converted equally well by IMP-6, IMP-1, and G262A, but even more efficiently by the other mutants, and type II (ceftazidime, benzylpenicillin, ampicillin, and imipenem), which are hydrolyzed much less efficiently by all the mutants. G262V, S121G, F218Y, and F218I improve conversion of type I substrates, whereas G262A and IMP-1 improve conversion of type II substrates, indicating two distinct evolutionary adaptations from IMP-6. Substrate structure may explain the catalytic efficiencies observed. Type I substrates have R2 electron donors, which may stabilize the substrate intermediate in the binding pocket. In contrast, the absence of these stabilizing interactions with type II substrates may result in poor conversion. This observation may assist future drug design. As the G262A and F218Y mutants confer effective resistance to Escherichia coli BL21(DE3) cells (high minimal inhibitory concentrations), they are likely to evolve naturally.
β-Lactamases hydrolyze β-lactam antibiotics and thereby allow survival of pathogenic bacteria challenged by treatment with these agents. Metallo-β-lactamases (MBLs), also known as class B β-lactamases (Ambler 1980), contain one or two zinc ions and are important components of this antimicrobial defense mechanism. They have become a severe clinical problem due to their broad substrate spectra and potential for horizontal transference (Laraki et al. 1999; Lauretti et al. 1999; Franceschini et al. 2000; Iyobe et al. 2000; Livermore and Woodford 2000).
Class B has been further divided into the three subclasses, B1 to B3, based on primary structure, and a general numbering scheme has been suggested (Galleni et al. 2001) which will be used throughout this paper. Subclass B1 is the most intensely investigated, and the structures of four of its members have been solved, either as free enzymes or enzyme-inhibitor complexes (Carfi et al. 1995; Concha et al. 1996, 2000; Garcia-Saez et al. 2003). A detailed catalytic mechanism has been proposed for the hydrolysis of nitrocefin (NIT) by CcrA, a binuclear zinc enzyme from Bacteroides fragilis (Wang et al. 1999). A zinc-bound hydroxide acts as a nucleophile and attacks the carbonyl carbon of the β-lactam. Cleavage of the amide bond yields an anionic intermediate, which is stabilized by coordination of the resulting carboxylate to Zn1 and the anionic nitrogen to Zn2. The anionic nitrogen is then protonated to form the product; the protonation is the rate-limiting step in the catalytic cycle. Mutational analyses (Yang et al. 1999; Haruta et al. 2000, 2001; Yanchak et al. 2000; Materon and Palzkill 2001; Carenbauer et al. 2002; de Seny et al. 2002; Huntley et al. 2003; Hall 2004), NMR studies (Scrofani et al. 1999; Huntley et al. 2000, 2003), and molecular modeling (Salsbury et al. 2001; Antony et al. 2002; Suarez et al. 2002a,b; Oelschlaeger et al. 2003a,b) have provided additional information on the structure and dynamics of these binuclear MBLs.
It is not clear whether all substrates form an anionic intermediate as NIT (Fast et al. 2001; Moali et al. 2003). However, based on the following observations, a role of the anionic intermediate for other substrates seems reasonable:
Benzylpenicillin (PEN) and cephaloridine (LOR) were converted less efficiently by an engineered mononuclear mutant of the binuclear CcrA (kcat
were 6% and 5% of the wild-type kcat
). This decrease in catalytic efficiency is similar to that observed with NIT (kcat
was 3% of the wild-type kcat
) (Fast et al. 2001
). The most likely way that the binuclear enzyme increases kcat
compared to the mononuclear enzyme is by stabilizing the intermediate and thus decreasing the energy barrier of the overall reaction.
We were able to model the binuclear imipenemases IMP-1 and IMP-6 in complex with anionic intermediates of the four cephalosporins cephalothin (CEF), cefotaxime (CTX), LOR, and ceftazidime (CAZ) in unconstrained molecular dynamics (MD) simulations (Oelschlaeger et al. 2003b
Although the evolutionary pathways that led to improved catalytic efficiency toward β-lactam antibiotics among binuclear enzymes are not completely clear, valuable insights can be obtained by comparing the substrate profiles of existing MBL variants, e.g., the imipenemases IMP-1 to IMP-13 (Docquier et al. 2003; Toleman et al. 2003). Thus, Iyobe et al. (2000) proposed that IMP-3 evolved into the more efficient enzyme IMP-1 via only two mutations. It has also been suggested that merely changing residue 262 of IMP-6 from glycine to serine (IMP-1) stabilizes the anionic intermediate of certain β-lactam substrates bound to the protein, thus enhancing catalysis (Oelschlaeger et al. 2003b).
In addition to naturally occurring variants, artificial mutants can reveal alternate evolutionary pathways to improved catalytic efficiency. Directed evolution methods have been applied to screen for improved serine β-lactamases (Orencia et al. 2001; Voigt et al. 2002) and MBLs (Ponsard et al. 2001; Hall 2004). A method combining computational and experimental screening has been used to generate TEM-1 (class A) variants with increased resistance (Hayes et al. 2002). Focusing on conserved positions in the active site, a mutational study of IMP-1 identified residues essential for efficient zinc binding (Haruta et al. 2000), which was confirmed by crystallography (Concha et al. 2000): Zn1 is coordinated by H116, H118, and H196, and Zn2 is coordinated by D120, C221, and H263. Also, conserved residues were tested for their role in substrate binding: While K224 was found to be important, N233 could be mutated without significant loss of activity (Haruta et al. 2001). These findings were supported by MD simulations (Oelschlaeger et al. 2003a). Materon and Palzkill (2001) took these studies further and randomized IMP-1 codons to create a library that allowed all possible amino acid substitutions at all positions in and near the active site. In addition to confirming essential residues, this approach revealed that many other positions in the active site could tolerate amino acid substitutions; the N233A mutant even converted the investigated substrates more efficiently. Using site-directed mutagenesis, Moali et al. (2003) recently reported that the importance of a flexible loop covering the active site depends on the nature of the substrate.
While previous reports focused on the active site, we explored the impact of remote mutations on substrate specificity. The G262S point mutation distinguishing IMP-1 from IMP-6 results in significantly improved catalytic efficiency toward LOR, CAZ, PEN, ampicillin (AMP), and imipenem (IMP) (Iyobe et al. 2000). This change in specificity occurs even though S262 does not ligate the zinc ions and is not in contact with the substrate. MD simulations suggest that this effect occurs indirectly via a domino effect in which the neighboring H263 is rendered less flexible, thus stabilizing the enzyme-substrate intermediate complex for these substrates and enhancing catalytic activity (Oelschlaeger et al. 2003b). In the present study, we explored this possibility further by determining the impact of other point mutations in the vicinity of position 262 that are also remote from the active site. We restricted mutants to those that could occur via single-nucleotide substitutions, thus focusing on IMP-6 variants that can evolve naturally. All mutants showed altered substrate spectra, some exhibiting significantly improved catalytic efficiencies toward certain substrates. Possible mechanisms for these observations are discussed.