Miriam Hood
Theobald Laboratory
Department of Biochemistry
Brandeis University
Active Site Dynamics of a Bifunctional Apicomplexan Malate and Lactate Dehydrogenase Ancestral Intermediate
Poster Abstract
A variety of human diseases, including malaria, are caused by unicellular eukaryotes of the phylum Apicomplexa, which have evolved a highly specific lactate dehydrogenase (LDH) from malate dehydrogenase (MDH), which may be a potential drug target. A putative promiscuous intermediate in the evolutionary path makes Apicomplexan LDHs and MDHs an excellent model for studying the role of such intermediates in the evolution of specificity.
A six-amino acid insertion conferred pyruvate activity in LDHs, by shifting the key catalytic residue from an arginine in MDHs to a tryptophan. There are two hypothesized conformations for the active site of the bifunctional ancestor intermediate, but only an LDH conformation is seen in X-ray crystallography.
Heteronuclear single quantum coherence nuclear magnetic resonance (HSQC NMR) spectroscopy was attempted to visualize the enzyme in the presence of each substrate, but was unsuccessful due to the size of the tetramer formed. Disruptive mutations were used to create a stable dimer, small enough to produce a well-resolved HSQC NMR spectrum. HSQC NMR spectra of the dimer with oxaloacetate and pyruvate are well resolved, but do not show significant peak shifts.
Personal Statement
This summer marks my third and final summer as an undergraduate researcher in the Theobald Lab. I can say, without a doubt, that my time in the lab has been one of the most transformative experiences of my time in college. As an incoming first-year student, I knew I wanted to participate in undergraduate research, but I had no idea of just how involved I would become and how much I would come to enjoy and appreciate engaging in active fields of scientific research. In the Theobald Lab, I have come to see evolution in an entirely new light, one that is far more complex and deeper than the initial theory introduced in high school biology. I have had the opportunity to apply the method and theories discussed in my biochemistry courses to biologically relevant enzymes, dig further into scientific papers, and have meaningful conversations with graduate students and professors about the value of basic scientific research. Summer research, in particular, has be incredibly important to my time in the lab, allowing me to focus fully on my project for several intense and productive weeks.
The M. R. Bauer Foundation Fellowship has allowed me to continue my research on a bifunctional Apicomplexan malate and lactate dehydrogenase ancestral intermediate for the combined Bachelor of Science and Master of Science degrees in biochemistry. Over the course of this summer I have been able to fully characterize the kinetics of this ancestral intermediate, determine melting point data for the intermediate and related ancestors, and begin using new techniques, such as x-ray crystallography and fluorescence spectroscopy. Conducting this research has also allowed me to attend and present a poster at the annual Society for Molecular Biology and Evolution meeting. Attending the event and presenting a poster provided me with an opportunity to expand my presentation skills. The meeting also allowed me to discover just how broad and far reaching the field of molecular evolution is, and engage with other scientists who use a wide variety of unique approaches to the field. My time in the lab supported by the M.R. Bauer Fellowship, has not only facilitated the progress of my research, but has given me the time to build a strong foundation for a future in scientific research.