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Evolutionary relationships in proteins structure and function, Proteomic analysis of metabolic enzymes in plants and bacteria.
Research in my laboratory focuses on elucidation of functional and structural domains of various evolutionarily conserved enzyme families, and studies of hydroxyacid and enoate metabolism in bacteria and plants. This is done by a combination of molecular biological and chemical/structural approaches. Much of the research utilizes proteomic methods, and purified recombinant proteins that can be easily manipulated by site-directed mutagenesis, chemical modification, and mass spectral analysis. Our current projects described below:
Characterization of the Beta-Hydroxyacid Dehydrogenases:
The beta-hydroxyacid dehydrogenases are a family of structurally and mechanistically related enzymes. All of these enzymes catalyze the reversible oxidation of beta-hydroxyacid substrates with differences in their substrate and dinucleotide cofactor specificities. These enzymes have an evolutionarily conserved functional domain structure reflected in an amino acid sequence homology, which is very characteristic of this family. Site-directed mutagenesis and site-specific chemical modification experiments have revealed the functions of each of the conserved domains. We have recently identified pyrroline-5-carboxylate reductase as an evolutionarily distant member of this family. Mutagenesis and chemical modification experiments are planned to map out specific structural elements important to the catalytic activity of this enzyme.
Characterization of Bacterial Aldarate Isomerases:
Genes coding for several isoforms of hydroxypyruvate isomerase are present in the genomes of many bacteria. These enzymes likely play important catalytic roles in bacterial metabolic pathways; however, it is not clear what pathways these are. Very little is known about how these enzymes work, and it is not clear why there are multiple isoforms. We have cloned both isoforms from E. coli and are comparing their substrate-specificities and kinetic properties. Site-directed mutagenesis and site-specific chemical modification experiments will also be used to map specific structural elements important to the catalytic activity of this family of enzymes.
Proteomic Analysis of Hydroxyacid and Enoate Metabolism in Bacteria:
The genomes of many bacteria code for multiple homologues of the beta-hydroxyacid dehydrogenases. In some cases the metabolic function of specific isoforms are clear. For some homologues, little is known about their exact biological functions. We are using genomic and proteomic approaches to determine the functions of several beta-hydroxyacid dehydrogenases in E. coli and H. influenzae. Our proteomic approach utilizes a combination of 2-D gel electrophoresis and capillary HPLC techniques as well as a number of "targeted proteomic" methods developed in our laboratory. We are also using this approach to study the metabolism of specific enoates like acrylate and sorbate in E. coli.
Proteomic and Genomic Analysis of Hydroxyacid and Enoate Metabolism in Plants:
Plant genomes contain a surprising multiplicity of genes coding for beta-hydroxyacyl-CoA hydrolases and beta-hydroxyacid dehydrogenases. These enzymes appear to be distributed in different plant tissues and even in different organelles where they likely play important roles in enoate metabolism and hydroxyacid metabolism. Clues to the biological function of these enzymes are gained by cloning and expressing them as recombinant enzymes which can be used to study their enzymological properties. Several genomic and proteomic approaches are also planned to study the expression of specific enzyme forms in plant tissues under various environmental conditions that may affect their metabolic functions.
Recent Publications
J. W. Hawes, K. L. Knudtson, H. Escobar,
G. S. Grills, T. C. Hunter, E. Jackson-Machelski, H. Lin, D. S. Needleman,
R. Pershad, T. W. Thannhauser, and G. J. Wiebe, “Evaluation of Methods for
Sequence Analysis of Highly Repetitive DNA Templates”, J. Biomol. Tech.,
17, 138-144 (2006).
J. H. Dominguez, P. Wu,
J. W. Hawes, M. Deeg, J. Walsh, S. C. Packer, M. Nagase, C. Temm, E. Goss,
and R. Peterson, “Renal Injury: Similarities and Differences in Male and
Female Rats with the Metabolic Syndrome”, Kidney Int., 69(11),
1969-1976 (2006).
A. Srirangam, R. Mitra,
M. Wang, J. C. Gorski, S. Badve, L. Baldridge, J. Hamilton, H. Kishimoto, J.
W. Hawes, L. Li, C. M. Orschell, E. F. Srour, J. S. Blum, D. Donner, G. W.
Sledge, H. Nakshatri, and D. A. Potter, “Effects of HIV Protease Inhibitor
Ritonavir on Akt-regulated Cell Proliferation in Breast Cancer”, Clin.
Cancer Res., 12(6), 1883-1896 (2006). |
