Research Areas

The overriding goal of research in the Loewen lab has been to gain a better understanding of the oxidative stress response. The methods used to achieve this goal changed over the years with the primary focus eventually being on 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 were the main objects of intense and detailed study.

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.

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 30. 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.

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. Subsequently, the binding site for INH was identified leading to a revised mechanism for how the pro-drug is converted to isonicotinyl-NAD, its active form. Significant insights into the catalatic reaction mechanism of KatGs have appeared in the past few years.

Now that my lab is essentially shut down, I must in closing recognize that none of this work would have been possible without many collaborations including, in particular, those in crystallography with Ignacio Fita (Barcelona), in Electron Paramagnetic Resonance with Anabella Ivancich (Saclay), and in computation with Carme Rovira (Barcelona) and Miguel Machuqueiro (Lisbon).

H. pylori catalase HPC

B. pseudomallei catalase-peroxidase KatG