Research Areas
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The overriding goal of research in the Loewen lab is to gain a better understanding of the oxidative stress response. The methods being used to achieve this goal have changed over the years and the primary focus now lies in the catalytic mechanisms used by catalases to destroy hydrogen peroxide before it damages cellular components. Of the three main classes of catalases, the monofunctional heme catalases and bifunctional catalase-peroxidases are the objects of intense and detailed study. Background The importance of catalases to life is evident in the fact that most organisms produce more than one, with three or four not being uncommon. For example, the common bacterium Escherichia coli produces two catalases or hydroperoxidases, HPI and HPII, and these two enzymes have served as model systems for studies of catalase structure and function in the Loewen laboratory for almost 30 years. HPI is a member of the bifunctional catalase-peroxidase family and is active as both a dimer and a tetramer of identical 81 kDa subunits, each containing one heme b group. HPII is a monofunctional, large subunit catalase, which is active as a tetramer of identical 84 kDa subunits, each with a heme d group. Much of the work describing the characterization, genetics and molecular genetics of catalases from E. coli has originated in the Loewen lab. The genes encoding HPI (katG) and HPII (katE) were mapped, cloned and sequenced in the 1980s. The demonstration that HPII expression was controlled by KatF lead to the characterization of KatF as an alternate sigma transcription factor controlling stationary phase gene expression. KatF was ultimately renamed RpoS or Sigma-S and work in other laboratories went on to characterize the RpoS regulon. Catalases HPII became the focus of an detailed structure function study that included the construction and characterization of over 100 mutants and the structural refinement of 12. Important insights into the catalytic mechanism have been gained of which one highlight is the involvement of an electrical potential field in the entrance channel that orients incoming substrate as it approaches the deeply buried active site. In addition, structures of catalases from the plant pathogen Pseudomonas syringae, CatF, and the human pathogen Helicobacter pylori, HPC, have been determined, providing insights into the diversity of catalase properties. Catalase-peroxidases The catalase-peroxidase HPI (or KatG) of E. coli is very similar to KatG from Mycobacterium tuberculosis which activates the widely used anti-tubercular pro-drug isoniazid (INH). Interest in KatGs increased immensely when it was found that the major cause of the spread of isoniazid resistance tuberculosis in the world was the mutation of the katG gene in M. tuberculosis. HPI from E. coli proved to be not amenable to crystallization despite 15 years of trying, but in 2003 the crystallization and structure refinement of KatG from Burkholderia pseudomallei was achieved in collaboration with Ignacio Fita (Barcelona, Spain). This has provided a number unique and exciting insights into the catalytic mechanism of the enzyme. One particular highlight was the demonstration of a “molecular switch” role for Arg426 which is involved in modulating the catalase reaction. A second discovery was of an NADH oxidase activity in KatGs that has implications in the mechanism of INH activation. Future directions Work is continuing in both classes of enzymes. In KatGs, the immediate goal is to integrate the apparently disparate mechanisms surrounding the active site that are essential for imparting catalase activity to what is essentially a peroxidase. There is such a level of complexity in the enzyme that the basic kinetic characterization of variants is being expanded through the application of other approaches including collaborations in crystallography with Ignacio Fita (Barcelona), in Electron Paramagnetic Resonance with Anabella Ivancich (Saclay), in absorbance spectroscopy with Andy Smith (Sussex) and Christian Obinger (Vienna), and in quantum mechanical calculations with Carme Rovira (Barcelona). In monofunctional catalases, many questions similar to those in KatGs remain, including the existence and location of protein radicals in the oxo/hydroxferryl heme reaction intermediates and the roles of alternate channels leading to the active site. |
 H. pylori catalase HPC 
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