Steve Ealick's Research Group

Projects


We use X-ray crystallography to study the three-dimensional structures of proteins. The structural information is used for drug design, protein engineering, to understand catalytic mechanisms, and to explore protein evolution. Our group is also involved in the development of tools and techniques associated with synchrotron radiation, especially multiple wavelength anomalous diffraction (MAD), single wavelength anomalous diffraction (SAD) and microdiffraction. Our main projects include studies of enzymes involved in purine nucleotide metabolism and pyrimidine nucleotide metabolism, studies of enzymes involved in cofactor biosynthesis, especially thiamin biosynthesis and pyridoxal 5'-phosphate PLP biosynthesis and enzymes involved in polyamine biosynthesis, purine biosynthesis, pyrimidine biosynthesis, and diphthamide biosynthesis.

Because of the role of purine and pyrimidine nucleotide metabolism in diseases such as cancer and viral infection, many of the enzymes involved are targets for drug design. We have focused considerable effort on nucleoside phosphorylases, which are found in both purine and pyrimidine pathways. Purine nucleoside phosphorylase is required for T-cell development. In collaboration with Professor Eric Sorscher, University of Alabama at Birmingham School of Medicine and Dr. William Parker, Southern Research Institute, we also study bacterial purine nucleoside phosphorylase because of its application in prodrug activation in the context of gene therapy.

We are investigating enzymes involved pathways that synthesize, degrade or utilize purines and pyrimidines. Systematic investigation of an entire biochemical pathway provides important clues about protein evolution. Our current emphasis is on multifunctional enzymes associated with these pathways that had not been previously characterized. We are also particularly interested in the novel activities associated with two newly discovered catabolic pathways: one for each pyrimidines and one for purines. Purine utilization is initiated by a cyclohydrolase reaction. Because many uncharacterized bacterial operons contain cyclohydrolase genes, we are exploring novel biosynthetic pathways for purine derived metabolites. Our primary collaborator in these studies is Professor Tadhg Begley of Texas A&M.

We are also interested in cofactor biosynthesis. We are currently focusing our efforts in this area on thiamin and pyridoxal 5'-phosphate (PLP) biosynthesis. Many of the reactions catalyzed by the enzymes in thiamin biosynthesis involve unprecedented chemistry and elucidation of catalytic mechanism is a major goal. By studying these enzymes, we have also discovered interesting evolutionary links to other pathways. PLP is the biologically active form of vitamin B6 and is an important cofactor for several of the enzymes involved in the metabolism of amine-containing natural products such as amino acids and amino-sugars. Of particular evolutionary interest is the finding that there are two distinct PLP biosynthetic pathways that have not yet been found to coexist in the same organism. Our primary collaborators for these studies are Professor Tadhg Begley of Texas A&M and Dr. Ivo Tews of the University of Southampton.

Polyamines have been implicated in many biological processes. Because of their alternating positive and hydrophobic regions, polyamines are able to bind to protein and nucleic acids in unique ways. Production of polyamines is highly regulated and is correlated with the cell cycle. We study enzymes of polyamine biosynthesis in collaboration with Dr. Wayne Guida of the University of South Florida and the Moffitt Cancer Center at USF, and Dr. John Secrist III, Dr. William Waud of the Southern Research Institute and Margaret Phillips of the University of Texas Southwestern Medical Center. We plan to use the structures of these enzymes to design novel anticancer and antiparasitic drugs.

A recent emphasis is the study of enzymes involved in diphthamide biosynthesis. Diphthamide is a unique posttranslational modification that occurs in all eukaryotes. The biosynthesis of diphthamide requires multiple proteins, and mutations in several of them have been connected to cancer. We plan to study the mechanism of diphthamide biosynthesis and the function of each protein required for the biosynthesis. This will provide important insights into the function of diphthamide, the regulation of diphthamide biosynthesis, and the mechanism of tumor formation in the absence of diphthamide biosynthesis, possibly leading to new ways to treat or prevent cancer. Our primary collaborator for this work is Dr. Hening Lin of Cornell University.

Finally, we are interested in synchrotron radiation and its application in macromolecular crystallography. Our group lead an effort to construct, and is now operating a facility at the Advanced Photon Source, that is designed for the study technically challenging crystallographic samples. The Northeastern Collaborative Access Team (NE-CAT), with Professor Ealick as director, is operating undulator beamlines at Sector 24 of the Advanced Photon Source (APS) at Argonne National Laboratory in Argonne, Illinois. Of particular interest to the crystallographic community are the microdiffractometers that we have deployed for analyzing crystals that are much smaller than those usually usable and the robotics interface that makes data collection more streamlined. We installed a pixel array detector on the beamline and are making increasing use of remote data collection for the convenience of our users.



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