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Metals in Biological Systems  
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RESEARCH
 
Our research mainly centres on elucidating various aspects of the structure-function relationship of metalloproteins. In this regard, we are currently focusing on two zinc-containing enzymes, the anthrax lethal factor (LF) and certain members of the metallo-b-lactamase family. In addition, we are interested in developing simple (yet powerful) analytical methods for the quantification of metal ions (particularly those bound to metalloproteins) and other analytes.
 
 
Anthrax Lethal Factor (A mononuclear zinc endopeptidase)
 
Anthrax is an infectious disease caused by the rod-shaped, Gram-positive bacterium Bacillus anthracis. The disease mainly affects animals (mainly cattle and other herbivores), but can occasionally be transferred to humans (click here for a 'Brief History of Anthrax' and its use as a biological weapon). The toxic effects associated with anthrax are mainly a consequence of the secretion of three proteins which constitute the anthrax toxin. These are: the protective antigen (PA), the lethal factor (LF), and the edema factor (EF). The protective antigen, so-called for its use in vaccines, is responsible for the translocation of LF and EF into the host cell cytosol where these proteins function enzymatically.
 
LF is a zinc-dependent metallo-endopeptidase (90 kDa), which catalyzes the cleavage of mitogen-activated protein kinase kinases (MAPKKs) within their N-termini. Proteolysis of MAPKKs leads to a disruption of major cell signaling pathways (with deadly consequences). LF contains four distinct domains. While domain 1 is responsible for binding to PA during translocation, domains 2 to 4 are involved in substrate recognition and binding (see Figure). Furthermore, domain 4 harbours the active site of LF, i.e. the site where the target substrates (MAPKKs) are cleaved near their N-termini. The 'heart' of the active site of LF is comprised of a single Zn(II) ion, which is tetrahedrally coordinated by three amino acid residues (His686, His690, Glu735) and a single water molecule (or hydroxide ion). The Zn(II) ion activates the water/hydroxide ligand for nucleophilic attack on the substrate peptide bond to be cleaved. A glutamate and a tyrosine residue are also thought to be involved in the catalytic cycle. While Glu687 may act as a general base, Tyr728 appears to function as a general acid (protonation of the amine leaving group).
  LF crystal structure
 
Crystal structure of LF. The zinc ion is depicted in magenta.
 
Our research relating to LF centers on elucidating the metal requirement and mechanism of the enzyme. For instance, we are investigating whether the enzyme is only active with zinc, or whether (and how) other metals can functionally substitute for zinc. Furthermore, we are interested in the role that certain amino acids in or near the active site play in the overall catalytic mechanism. In addition to these mechanistic studies, we are also interested in the development of novel inhibitors of the enzyme. Finally, we are currently probing the folding and unfolding behaviour of LF. LF is known to partially unfold during the PA-mediated translocation into the cytosol. It is completely unclear at the present time whether the zinc ion is retained during these unfolding events.
 
Metallo-b-Lactamases (Binuclear zinc enzymes)
 
b-Lactamases are enzymes which are capable of deactivating b-lactam antibiotics by hydrolysis of their endocyclic amide bond. Thus, these enzymes can be regarded as one of the major contributors to the emergence of microbial resistance to b-lactam antibiotics. There are four distinct classes of b-lactamases (designated A to D). While enzymes of the A, C, and D families employ a serine residue in their catalytic mechanism, class B enzymes (termed metallo-b-lactamases, MBLs) are zinc-dependent metalloproteins requiring one or two zinc ions for their function. In recent years, MBLs have received considerable attention since they hydrolyze most b-lactams, including penicillins, cephalosporins, clavulanic acid, penicillanic acid sulfones and carbapenems. Most MBLs are chromosomally encoded, but some are of plasmidic origin (e.g., IMP and VIM enzymes). This feature is a matter of concern since the plasmids harbouring a MBL-encoding gene can potentially be transferred to other (more pathogenic) microorganisms.
 
In collaboration with multiple research groups in Canada and the UK, our group is currently involved in investigating certain types of compounds for their inhibitory effect (potency and mechanism) on these enzymes. In addition, we are investigating the role of each of the two metal ions in the catalytic mechanism of MBLs.
 
Development of Analytical Techniques
 
We are interested in developing simple and cost-effective analytical techniques for the determination of metal ions in biological samples (in particular metalloproteins) using simple chromophoric chelators. In addition, we are interested in using these compounds to determine more than one metal in the same sample. This is important when quantifying the metals in binuclear enzymes (e.g. Cu/Zn superoxide dismutase, Zn/Co hybrids of binuclear metallohydrolases, metal-substituted enzymes, etc.). Apart from analyzing metals, we are also working towards the development of ultrasensitive fluorescent (metal-based) chemosensors for a variety of analytes including cyanide, sulfide and fluoride.