Research Interests
My research interests involve the mechanistic and structural characterization of metalloenzymes using spectroscopic, molecular biological, and kinetic techniques. The enzyme systems under study are biomedically-relevant; in several cases, the ultimate goal is the rational design and preparation of a clinically-useful inhibitor. We are taking a multidisciplinary approach, using bioinorganic, bioorganic, and physical studies to develop compounds that will specifically inhibit important biomedical targets.
The overuse of antibiotics in the clinical setting has resulted in a large number of bacteria which are resistant to most or all commonly-used antibiotics. These bacteria have become resistant to the antibiotics in a number of ways, such as producing enzymes which destroy the antibiotic, producing proteins which bind to and transport the antibiotic out of the cell, or developing alternative metabolic pathways which would allow for the bacterium to survive in the presence of the antibiotics. Currently, the most common and most often prescribed antibiotics are b-lactam-containing compounds, which include the penicillin, cephalosporin, cephamycin, and carbapenem families. The b-lactam-containing antibiotics exhibit their antimicrobial activity by inactivating bacterial transpeptidases, which are enzymes required for bacterial cell wall synthesis. The inactivation of transpeptidase results in a weakened bacterial cell wall which is susceptible to osmotic lysis.
The most common way for bacteria to become resistant to b-lactam-containing antibiotics is to produce an enzyme called b-lactamase. b-Lactamases hydrolyze and inactivate b-lactam-containing antibiotics. There are over 200 different b-lactamases, and fortunately, there are clinically-useful inhibitors that can be used against most of them. However, one class of b-lactamases, metallo-b-lactamases, have been shown to bind 1-2 Zn(II) ions per monomer, to hydrolyze all tested b-lactam-containing antibiotics, and to be produced by several pathogenic organisms. Significantly, there are no known clinically-useful inhibitors of the metallo-b-lactamases. Recent studies have demonstrated significant structural and mechanistic differences among the metallo-b-lactamases, and previous inhibition studies have suggested that one inhibitor may not inhibit all metallo-b-lactamases.
In my lab, we are currently conducting structural and mechanistic studies on the metallo-b-lactamases from S. maltophilia (L1), A. sobria (imiS), and B. fragilis (CcrA) in an attempt to uncover mechanistic and structural similarities between the enzymes. These similarities will be targeted for the preparation and synthesis of potential inhibitors.
Zn(II) is an essential trace element of many proteins and is required for life in all organisms. In fact, Zn(II) serves as a catalytic cofactor in members of all six major functional classes of enzymes and a structural cofactor for many other proteins. Nonetheless, high levels of Zn(II) are TOXIC to cells, presumably due to Zn(II) binding to adventitious metal binding sites and to binding sites that are supposed to be occupied by other metal ions. In addition, the Crowder lab has shown that too little Zn(II) is bacteriostatic (Wenzel and Crowder, unpublished), meaning that bacterial cells cannot grow without Zn(II). Therefore, it is essential that cells maintain homeostatic mechanisms that regulate absorption, distribution, cellular uptake, and excretion of Zn(II).
Pioneering work by O'Halloran and others have
revealed that cells have an optimal zinc quota; for example, E. coli cells have
zinc quota of ca. 2 X 105 atoms per cell (millimolar
concentrations). However, studies by O'Halloran have
indicated that essentially all of this Zn(II) is bound
by proteins or is stored in vesicles and that there is no persistent pool of
free Zn(II) inside an E. coli cell. By using a cell assay and a Zn(II) regulatory protein, O'Halloran
and coworkers estimated that there is much less than one free Zn(II) ion in an
E. coli cell!!!! Clearly, the levels of Zn(II) inside
the cell are strictly regulated, and many cell membrane-associated proteins
that pump Zn(II) into or out of cells have been identified in bacterial and
eukaryotic organisms.
However, these results pose some significant questions about Zn(II) transport within the cell. First, what are the proteins that bind all of these Zn(II) ions? It has been estimated that at least 12% of the total Zn(II) in E. coli is bound by only 8 proteins and that RNA polymerase and five tRNA synthetases tie up most of this Zn(II). There are at least 40 other proteins in E. coli that tightly bind Zn(II), and there are potentially an infinite number of non-specific, weak binding sites for Zn(II) provided by proteins, nucleic acids, and amino acids. A second question arises: with all of these potential Zn(II) sites inside E. coli, how does Zn(II) get delivered to the correct proteins? Third, how is the Zn(II), which is brought in by the influx pumps, transported to the storage vesicles, to efflux pumps, or to Zn(II) metalloproteins inside the cell? Recently, several groups have suggested that metallothionein is a Zn(II) metallochaperone; a protein that can transport Zn(II) within the cell. While this is possible, metallothionein can bind a number of different metal ions in vivo, presenting a problem of loading the wrong metal ion into proteins as they are being synthesized and of transporting the incorrect metal ion to storage vesicles or to efflux pumps. Another potential problem with metallothionein being the sole Zn(II) metallochaperone is that Zn(II) binds too tightly to metallothionein. The exchange of Zn(II) from metallothionein to other Zn(II) binding proteins that have much weaker Zn(II) binding would be thermodynamically difficult if not impossible. While it is possible that loading of Zn(II) into some Zn(II) binding proteins may be under kinetic control, Zn(II) loading to some proteins appears to be under thermodynamic control. To address the questions posed by these studies, O'Halloran and others have predicted that there are, as yet unidentified, Zn(II) metallochaperones that transport Zn(II) within the cell. This prediction stemmed from studies on the Cu(II) metallochaperones that have been identified recently. The existence of Zn(II) metallochaperones would also help to explain how Zn(II) is incorporated into proteins as they are being synthesized.
The main goals of this study are to identify the zinc (Zn(II))
metallochaperones in E. coli by utilizing modern
protein and nucleic acid profiling techniques. Protein expression profiling
with 2D gel electrophoresis will be used to monitor which proteins in E. coli
are differentially produced in response to high and low levels of extracellular Zn(II). Mass spectrometry of spots on the
resulting gels will be used to identify the proteins. Since it is possible that
some proteins can be missed with 2D gels (particularly membrane bound or
associated proteins), nucleic acid profiling (gene arrays) will be used to
monitor the levels of mRNA in the same samples listed above. Future studies
will involve understanding how known Zn(II) transport
proteins interact with other proteins. In addition, any potential Zn(II) metallochaperones that are
identified will be cloned, over-expressed, purified, and characterized by using
metal binding assays and spectroscopic techniques.