Membranes are the essential barriers that define and delineate the inside versus the outside of a cell. Membranes are composed of fatty acids, and in contrast to humans, bacteria synthesize all of the needed fatty acids for their cellular membranes. Fatty acids are synthesized by a series of enzyme catalyzed chemical reactions. The enzyme that catalyzes the first reaction in fatty acid synthesis is acetyl-CoA carboxylase. Acetyl-CoA carboxylase is a multifunctional enzyme composed of two different enzymes: biotin carboxylase and carboxyltransferase. The first aim of this project involves trying to understand how the three-dimensional structure of biotin carboxylase allows the enzyme to catalyze the reaction. For instance, the enzyme is composed of two identical subunits where each subunit can in theory catalyze the reaction. The fundamental question is: if each subunit is capable of catalyzing the reaction why does it exist as a dimer? One hypothesis that is being tested is that the subunits are not independent but instead depend on each other by alternating their catalytic cycles. Similar to the up and down motion of a bicycle pedal, one subunit catalyzes a reaction while the other releases products. The second and third aims of the project involve trying to understand how the three-dimensional structure of carboxyltransferase participates in regulating the genes that code for the protein. In contrast to biotin carboxylase, carboxyltransferase is composed of two different subunits. The genes coding for these subunits are located at different positions in the bacterial chromosome yet the cell requires equal amounts of each subunit to make a functional enzyme. The fundamental question here is: how does a bacterium regulate expression of the genes coding for the subunits to obtain equal amounts? The carboxyltransferase molecule in bacteria contains a structural motif called a zinc finger that binds to RNA. The hypothesis is that carboxyltransferase regulates expression of both genes by binding to the RNA derived from those genes. When nutrients are low, carboxyltransferase binds to the RNA and inhibits protein synthesis. When nutrients are abundant and new membranes are needed, the substrate for the enzyme acetyl-CoA competes with RNA for binding and fatty acids are synthesized. Thus, carboxyltransferase appears to have two mutually exclusive "duel" functions: catalysis and translational regulation.
Broader Impacts of the Research: This project addresses fundamental questions in enzyme catalysis, subunit interactions, and regulation of translation in a model system. The eclectic approach will provide a detailed understanding of structure-function relationships that will have considerable impact on our view of the roles of the enzyme on the physiological level. This project will rely heavily on diverse graduate and undergraduate students to perform the experiments, serving as a training platform for the future scientific workforce using the eclectic tools of biochemistry and molecular biology. In addition, because the enzyme in this project is a carboxylase (using a form of carbon dioxide as substrate) the results of the research may have ramifications for engineering enzymes to reduce the carbon footprint in the environment. Lastly, because membranes are essential for life, acetyl-CoA carboxylase in plants is a logical target for herbicides. Understanding the structure and function of the enzyme can facilitate the design of more potent and environmentally safe herbicides.
In the previous 12 months 6 papers were published from this laboratory funded by NSF. Below is listed the publication followed by a short description of the findings and significance. The Three-Dimensional Structure of the Biotin Carboxylase-Biotin Carboxyl Carrier Protein Complex of E. coli Acetyl-CoA Carboxylase. Broussard, T.C., Kobe, M.J., Pakhomova, S., Neau, D.B., Price, A.E., Champion, T.S. and Waldrop, G.L. (2013). Structure21, 650-657. Determined the first three-dimensional model of the biotin carboxylase-biotin carboxyl carrier protein complex. This will aid in the development of new antibiotics and the development of biobased methods for the production of bulk chemicals. 2. Complex Formation and Regulation of Escherichia Coli Acetyl-CoA Carboxylase. Broussard, T.C., Price, A.E., LaBorde, S.M. and Waldrop, G.L. (2013). Biochemistry 52, 3346-3357. Determined the kinetic regulation of acetyl-CoA carboxylase and provided the first biochemical data showing how it is the regulated/rate-determining step in fatty acid biosynthesis. Again,this will aid in the development of new antibiotics and the development of biobased methods for the production of bulk chemicals. 3. Frontiers, Opportunities, and Challenges in Biochemical and Chemical Catalysis of CO2 Fixation. Appel, A.M., Bercaw, J.E., Bocarsly, A.B., Dobbek, H., Dupuis, M., DuBois, D.L., Ferry, J.G., Fujita, E., Hille, R., Kenis, P.J.A., Kerfeld, C.A., Morris, R.H., Peden, C.H.F., Portis, A.R., Ragsdale, S.W., Rauchfuss, T.B., Reek, J.N.H., Seefeldt, L.C., Thauer, R.K., Waldrop, G.L. (2013). Chem. Rev., 113, 6621-6658. This is a review of the various ways to fix CO2. A timely review that will hopefully help develop new methods to remove/decrease the every increasing levels of atmospheric CO2. 4. A Capillary Electrophoretic Assay for Acetyl Coenzyme A Carboxylase. Bryant, S.K., Waldrop, G.L. and Gilman, S.D. (2013). Analytical Biochem. 437, 32-38. Developed a non-radioactive assay for acetyl-CoA carboxylase using a capillary electrophoresis. This method can be used for basic research on this enzyme and also be used by pharmaceutical companies to find inhibitors that can be used as antibiotics. 5. Capillary Electrophoresis-Based Assay of Phosphofructokinase-1. Malina, A., Bryant, S.K., Chang, S.H., Waldrop, G.L. and Gilman, S.D. (2014). Analytical Biochem., 447, 1-5. Developed a non-radioactive assay for PFK using a capillary electrophoresis. This method can be used for basic research on this enzyme and also be used by pharmaceutical companies to find inhibitors that can be used as anti-obesity agents. 6. Computational Redesign of Bacterial Biotin Carboxylase Inhibitors Using Structure-Based Virtual Screening of Combinatorial Libraries. Brylinski, M. and Waldrop, G.L. (2014). Molecules, in press , . Used high performance computing to design new inhibitors of the enzyme biotin carboxylase which is a target for the development of new antibiotics. These molecules will provide a platform for pharmaceutical companies to develop broad spectrum antibiotics directed against biotin carboxylase.
