Prof. Robert P. Hausinger's lab

This laboratory explores specific aspects of microbial physiology and enzymology related to transition metals. One project in the laboratory involves a series of iron-containing enzymes that couple the oxidative decarboxylation of alpha-ketoglutarate (forming carbon dioxide and succinate) to the hydroxylation of specific primary substrates. The most exciting of these enzymes, AlkB, repairs DNA that is damaged by alkylating agents. Specifically, AlkB hydroxylates the deleterious methyl groups on 1-methyladenine or 3-methylcytosine to yield unstable intermediates that release formaldehyde and restore the undamaged bases. A second of these enzymes is TauD which decomposes taurine, a widely occuring sulfonate produced at high abundance in our cells. In three other enzymes of interest from a bioremediation standpoint, TfdA, RdpA, and SdpA hydroxylate the side chains of selected phenoxyalkanoic acids to produce the corresponding substituted phenols and glyoxylate or pyruvate. Our current efforts to characterize these proteins include (1) computational structure predictions and substrate docking analyses, (2) site-directed mutagenesis of active site residues, (3) steady-state kinetic studies with alternate substrates and inhibitors, and (4) transient kinetic analyses using spectroscopic approaches to examine reaction intermediates.

The second major emphasis in the laboratory focuses on the functions of a series of proteins (UreD, UreE, UreF, and UreG) that are responsible for activating the nickel-containing enzyme urease (comprised of UreA, UreB, and UreC). We have shown that UreE is a metallochaperone involved in delivery of nickel ions to the urease apoprotein when it is in complex with the other three accessory proteins. Nickel insertion occurs in a GTP-dependent manner, with UreG being the specific GTPase. Our current efforts are focused on (1) better understanding the specific protein:protein interactions required for this metallocenter assembly process, clarifying the kinetics of this activation process both in vitro and in vivo, and (3) elucidating the functions of the accessory proteins by a combination of computational structure prediction, site-directed mutagenesis, metal binding quantitation, and activity studies.

Undergraduate UBM participants working in this laboratory will work closely with a research associate or graduate student. They will choose one of our enzymes or activation proteins and learn basic biological techniques including cell growth, protein purification, possibly site-directed mutagenesis, and general kinetic characterization method. In addition, they will focus on developing expertise with a computational approach to analyze their protein such as predicting the protein structure, bioinformatic analysis of homologous enzymes, or simulating the transient kinetics of an enzyme. I have had numerous undergraduates become adept at protein purification / characterization and some have carried out site-directed mutagenesis. In addition, former undergraduates have contributed to analyzing steady-state and transient kinetics, including data simulations. Standard plotting programs such as Origin, SigmaPlot, and KaleidaGraph are used routinely in the lab, and our simulations have used programs that are packaged with the stopped-flow spectrophotometers as well as program C (developed at the University of Michigan by Professor Dave Ballou) and commercially available software. For the design of enzyme variants the students will utilize standard bioinformatic software such as Clustal W and 3-dimensional visualization tools such as SwissPDB viewer and Pymol. More advanced methods will be used for protein structure analyses that will be carried out in collaboration with various colleagues on campus (Drs. Feig, Wedemeyer, Kuhn, and Zavodszky), each of whom has interacted closely with undergraduates. The students will have the opportunity to learn methods such as Modeller to construct predicted structures and perhaps even some molecular dynamics approaches. Substrate binding modes will be explored by use of the program SLIDE, developed here at MSU.

Lab Home Page: http://www.mmg.msu.edu/95.html