Research

       Understanding the function of a macromolecule involved in a particular biological process often requires knowledge of its precise three-dimensional structure. This is especially relevant in cases where the molecule has a role in disease. Our group uses the technique of X-ray crystallography to determine high-resolution structures of macromolecules in a variety of systems. 

       Penicillin-binding proteins (PBPs) are the enzymes responsible for cell wall synthesis in bacteria and are the targets for the widely used ß-lactam class of antibiotics, including penicillin.  We have determined the structure of PBP 5 from E. coli to 1.9 Å resolution and have used this to probe the catalytic mechanism of PBPs and as a model system for the development of novel antibiotics.  In addition, we are studying a number of proteins from Neisseria gonorrhoeae (including penicillin-binding proteins) that are involved in peptidoglycan synthesis, interactions with host cell receptors or drug resistance.  The goals of this work are to understand the biological functions of these proteins, but also to assess their potential as new targets for therapeutic intervention.

       Pi-DNA complex - Replication of DNA is an essential process of all life forms.  Initiation of DNA replication in the E. coli R6K plasmid can occur from any of three origins, alpha, beta or gamma, and is therefore a useful model for the multi-origin replication system of eukaryotes.  R6K encodes a specific initiator protein called Pi that is the first to bind at the origin, eventually leading to replication fork assembly and elongation of the new DNA strands.  The protein also interacts with other proteins involved in the initiation process and, by a separate mechanism, represses its own translation.  Within the gamma origin of replication there are seven 22bp tandem repeats of the Pi-binding site termed iterons that require complete occupancy for initiation to occur.  To understand how the binding of Pi to these iterons triggers assembly of the replication fork, our goal is to determine the crystal structure of the Pi protein in complex with its iteron binding site.  This structure will show the precise role of Pi in the initiation process; specifically, whether the protein helps unwind DNA at the initiation site or whether it acts in a more passive manner by recruiting other proteins, such as DnaB helicase, to the replication fork.

       Phosphoglucose isomerase is a routine enzyme of glycolysis but also acts as a cytokine in a multitude of unexpected ways.  We have solved the structure of this enzyme from a variety of species and used these to probe the catalytic mechanism of the reaction.  Recently these investigations have extended to include novel PGIs from Archaeal species.  One such enzyme is a bifunctional phosphoglucose isomerase/phosphomannose isomerase from Pyrobaculum aerophilum, determined to 1.16 Å resolution, and another is a wholly novel PGI that belongs to the cupin family of proteins.  For the latter we have proposed a hydride shift mechanism of catalysis that is distinct from the proton transfer via a cis-enediolate intermediate that is the modus operandi of conventional PGI enzymes.  In addition to probing the enzymology of PGI, the long-term goal of this work is to identify and determine the structure of its cytokine receptor in mammalian organisms. 

 Other investigations in the laboratory include other aspects of DNA replication in bacteria and yeast systems, multifunctional enzymes, as well as RNA-protein interactions.