Robert P. Hausinger Research Interests
My laboratory explores specific
aspects of microbial physiology and enzymology related to transition metals.
For example, we study mechanisms of catalysis by metalloenzymes and characterize
the pathways for biosynthesis of protein metallocenters. We use an array
of experimental techniques and approaches that ranges from gene cloning
to enzyme kinetics, from site-directed mutagenesis to metal ion binding
assays, and from active site peptide studies to biophysical spectroscopic
methods.
One research focus in my lab centers on the nickel-dependent process of ureolysis catalyzed by the enzyme urease. We cloned the urease operon from Klebsiella aerogenes and can overexpress the genes such that 10% of the cellular protein is urease. The three-dimensional structure of this enzyme was determined by crystallographic methods and the dinuclear Ni center has been probed by a variety of spectroscopic methods. We are currently using site-directed mutagenesis methods to elucidate the catalytic mechanism of this enzyme. In addition, we are exploring the roles for four accessory proteins that function in urease metallocenter assembly. Urease characterization has important medical and agricultural implications. For example, bacterial urease is often associated with the formation of urinary deposits (kidney stones) during human infection, and uncontrolled hydrolysis of urea-based fertilizers can lead to crop damage. Thus, a detailed understanding of the urease mechanism and metallocenter assembly process may allow the design of pharmacologically or agriculturally effective inhibitors of this enzyme.
A second area of emphasis in the laboratory centers on the characterization of several ferrous ion and alpha-keto glutarate dependent hydroxylases. One of these enzymes, TfdA, catalyzes the initial step in mineralization of the herbicide 2,4-D (2,4-dichlorophenoxyacetic acid). A second enzyme, TauD, functions in bacterial metabolism of sulfonated compounds. Finally, the most recent project centers on AlkB, a unique DNA-repair enzyme. Current work with these enzymes includes metallocenter analysis by spectroscopic methods, characterization of site-directed mutant proteins and examination of alternate substrates and inhibitors.