Antibiotic resistance is increasingly becoming a problem in the control of infectious disease. Public health crises are emerging around the world as antibiotics fail to kill dangerous strains of bacteria. We are investigating resistance mechanisms to a family of antibiotics called beta-lactam antibiotics, which include penicillins, cephalosporins, and carbapenems.
Beta-lactam antibiotics remain the most useful chemotherapeutic agents in the fight against bacterial infections. Despite much progress in antibiotic design throughout the six decades since penicillin was introduced, resistance to beta-lactams is now a serious clinical problem, particularly in postsurgery, nosocomial infections and in immunosuppressed patients. The increasing use of these drugs in clinical settings places a highly selective pressure that favors pathogenic microorganisms through the development of various resistance mechanisms, the most prevalent of which is the generation of beta-lactamases—bacterial enzymes that aim to hydrolyze, and thus render inactive, these antibiotics. Since beta-lactamases were first clinically detected, there has been a need to design strategies to block their action.
There are two known types of beta-lactamases: serine-dependent beta-lactamases and metallo-beta-lactamases (MBLs). Serine beta-lactamases have been known for 50 years, and understanding their mechanism of action has led to the design of clinically useful inhibitors. By contrast, as a consequence of their unrivaled spectrum of activity and their resistance to therapeutic serine beta-lactamase inhibitors, MBLs are a clinical threat.
This project aims to analyze the structure of different MBLs and identify the mechanism they use to break down antibiotics. Several factors have so far precluded the design of a successful MBL inhibitor: (1) a lack of detailed knowledge of the mechanism of action of these enzymes; (2) the structural diversity displayed by MBLs from different sources, which hampers the discovery of common structural and mechanistic elements and thus the design of a pan-MBL inhibitor; and (3) a lack of information about the enzymes' evolutionary origin and how they may further evolve, which might help predict and disable future resistance mechanisms.
Mechanistic Studies on MBLs
The rate-determining step in beta-lactam hydrolysis involves a proton transfer. A conserved residue in all MBLs (Asp120) was originally proposed as the proton donor during catalysis. Mutagenesis of Asp120 and structural studies led us to discard this hypothesis. A computational study, conducted in collaboration with Paolo Carloni (Trieste, Italy) and Matteo Dal Peraro (University of Pennsylvania), led us to suggest that a water molecule bound to one of the zinc ions (called Zn2) plays the role of proton donor.
We engineered the active site of lactamases from other bacteria into BcII, which gave us two new chimeric enzymes in which either the location or the geometry of the Zn2 site was altered. Both mutants displayed noticeably impaired MBL activity. On the basis of the crystal structure of these mutants, we suggested that the orientation of the Zn2 site defines the catalytic performance of the enzyme.
We designed two further mutants in which one of the metal binding sites was abolished. In one mutant, we replaced the three Zn1 His ligands with Ser residues, and in the second one, we mutated the three Zn2 ligands (Asp120, Cys221, His263) to Ser. Thus, these mutants possessed only the Zn2- and the Zn1-binding sites, respectively. Even though the Zn1 site has been proposed to be responsible for most of the catalytic activity in BcII, both single-Zn mutants exhibited very poor hydrolytic capacity, suggesting that a di-zinc site is needed for catalysis.
By using rapid mixing techniques and various spectroscopic methods, we detected changes in the metal site of BcII during turnover in the millisecond timescale. We replaced the native zinc(II) ion with cobalt(II), giving rise to an active enzyme and providing a spectroscopic probe that allows us to monitor geometric changes in the active site during turnover. These experiments revealed that several enzyme forms coexist during turnover and that the metal ions adopt distinct geometries, depending on the substrate being hydrolyzed. Similar experiments with site-directed mutants have confirmed the role of conserved residues in the active site.
Structural and Functional Diversity within MBLs
We recently characterized a novel enzyme belonging to the family GOB, which is derived from a clinical isolate of the Gram-negative opportunistic pathogen Elizabethkingia meningoseptica. Even though it belongs to an already described subclass of MBLs, GOB displays two mutations on residues in its active site. In contrast to other closely related enzymes, GOB is fully active against a broad range of beta-lactam substrates using a single Zn(II) ion. Using a variety of spectroscopic techniques and enzymatic and mutagenesis experiments, we solved the structure of the metal-binding site. The single metal-binding site in GOB is essential for catalysis, even though a similar site in the di-zinc enzymes (the Zn2 site) has been regarded as noncatalytic. We propose that this metal site plays a role in favoring C-N bond cleavage in all MBLs, thus representing a common catalytic feature that could be targeted to design general MBL inhibitors.
In Vitro Evolution Experiments
We subjectedthe Bacillus cereus MBL (BcII) to a directed evolution scheme, which resulted in increased hydrolytic efficiency toward cephalexin. A systematic study of the hydrolytic profile, substrate binding, and active-site features of the evolved lactamase revealed that directed evolution shaped the active site by means of remote mutations, resulting in better hydrolysis of cephalosporins with small, uncharged C-3 substituents. One of these mutations is found in related enzymes from pathogenic bacteria and is responsible for the increase in that enzyme's hydrolytic profile. The mutations lowered the activation energy of the rate-limiting step instead of improving the affinity of the enzyme toward these substrates. We can thus draw the following conclusions: (1) MBLs are able to expand their substrate spectrum without sacrificing their inherent hydrolytic capabilities; (2) directed evolution is able to mimic mutations that occur in nature; (3) metal-ligand strength is tuned by second-shell mutations, thereby influencing catalytic efficiency; and (4) changes in the position of the second Zn(II) ion in MBLs affect substrate positioning in the active site. Overall, these results show that evolution of enzymatic catalysis can take place by remote mutations controlling reactivity and support the hypothesis that MBLs are still evolving in vivo.
Observing the X-ray structure of this mutant allowed us to assess the effect of each of these mutations on enzyme activity, structure, and stability. All these mutations, even though they were not at the active site, play a key role in tuning enzyme activity, and some of them are being selected by evolution in the clinical setting.
Clinical Detection of MBLs
Sensitive assays for specific detection of MBLs are increasingly in demand to prevent their dissemination and support infection control. We developed a novel microbiological assay using crude bacterial extracts to identify MBLs in nonfermentative Gram-negative clinical strains. We are pursuing this effort in collaboration with a network of public hospitals in Argentina; our aim is to provide a low-cost, rapid assay for early detection of MBL-based resistance in clinical strains.
Last updated March 2007
