My laboratory investigates microbial physiology and enzymology related to transition metals. In particular, we study mechanisms of catalysis by metalloenzymes and characterize the biosynthesis of protein metallocenters. We use an array of experimental techniques and approaches that includes gene cloning, site-directed mutagenesis, enzyme kinetics, metal ion binding assays, active site peptide studies, biophysical spectroscopic methods, and cellular assays for toxicity, DNA repair, and other processes.
One major emphasis in my laboratory focuses on characterization of lactate racemase, the most recently discovered nickel-containing enzyme. We demonstrated the enzyme contains a tethered niacin-derived pincer complex featuring a nickel-carbon bond (Science 349:66-69, 2015); this is the first example of a pincer complex in biology, contrasting with the extensive literature on synthetic pincer complexes by inorganic chemists. We continue to examine the mechanism and biosynthesis (requiring three helper proteins) of this unique cofactor, and are interested in its potential function in other enzymes.
A second area of emphasis centers on several ferrous ion and 2-oxoglutarate dependent hydroxylases (J. Biol. Chem. 290:20702-20711, 2015). TauD functions in the bacterial metabolism of sulfonated compounds and has become the paradigm of this enzyme family because of our spectroscopic, mutagenic and crystallographic studies. XanA is a fungal enzyme that metabolizes xanthine. A trypanosomal enzyme is required for the synthesis of base J in these protozoa. Other bacterial enzymes generate ethylene or metabolize glutaric acid. Finally, we study a mammalian enzyme that acts on methylated and AP sites in DNA. Current work with these fascinating enzymes includes metallocenter analysis by spectroscopic methods, characterization of site-directed mutant proteins, examination of alternate substrates and inhibitors, and cellular studies to assess functions.
A third research area studies the enzyme urease. Bacterial urease is 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 urease may allow the design of pharmacologically or agriculturally effective inhibitors of this enzyme. The conventional enzyme contains a unique dinuclear nickel active site and requires four "accessory proteins" to become active. One of the accessory proteins is a "metallochaperone" that delivers nickel ion. The other three accessory proteins work together to form a complex with urease apoprotein. We have been examining the role of these components in the mechanism of nickel incorporation into urease. In addition, we have recently studied a novel iron-containing form of urease. The structure of this protein closely mimics the nickel-dependent enzyme, but it uses a distinct mechanism for activation.