The biochemical importance of carbohydrates cannot be overstated for they are essential elements in nearly every physiological process and represent the most abundant biomolecules in living systems. Apart from their role in providing metabolic energy, carbohydrates are involved in a wide range of biological processes including the immune response, cell-cell interactions, fertilization, cell adhesion, and drug efficacy, among others. The focus of this research is on the unusual 2,3(4),6-trideoxyhexoses, which are found attached to important antibiotics such erythromycin A, antitumor agents such as adriamycin or insecticides such as spinosad. Specifically, this project is aimed at understanding the structure and function of the enzymes involved in the biosynthesis of D-forosamine and D-rubranitrose. These particular sugars decorate macrolide, or ansamycin aglycone scaffolds, respectively, and they were chosen for study because they contain novel functional groups and display strikingly different stereochemistries. Techniques to be utilized for this investigation include x-ray crystallography, site-directed mutagenesis, enzymatic synthesis of appropriate nucleotide-linked sugar ligands, and kinetic analyses. Ultimately, these investigations will reveal unprecedented chemistries and provide important and fundamental contributions to mechanistic enzymology. Broader Impacts: These projects were selected for study because they are ideally suited for instilling in middle school students the excitement of chemistry through biochemistry, which is absolutely critical for the continuation of high quality science in this country. Active collaborations between the Holden laboratory and Mr. Dan Toomey's science classes at Edgewood Campus Middle School in Wisconsin have already been established. During the first year of the project, Edgewood will select six qualified and interested students from the sixth, seventh, and eighth grades to begin an internship with a graduate student in the Holden laboratory. The ratio will be three middle school students to one graduate student. One day a week, each of these students will spend approximately three hours in the laboratory learning about the project and helping with the research. New sixth graders will be selected for the second year, while those students moving up to seventh and eighth grade will continue with the program. The primary benefits for the educational component of this research are threefold. First, students from grades six to eight will have hands on experience in a research laboratory working on biologically relevant projects. Second, the graduate students participating in this program will have the opportunity to teach in a laboratory setting and thus will already be on a trajectory toward establishing a meaningful and fulfilling career in education. Finally, the third benefit is to both Dan Toomey and the Holden laboratory. Dan Toomey has the educational background to know what is developmentally appropriate for the middle school students, and the Holden group has the scientific background to strengthen his curriculum. This synergy will have an enormous impact on how the topics of biochemistry and chemistry are presented to middle school students.
Natural products isolated from plants, bacteria, fungi, and many marine organisms have dramatically changed the course of human existence as is evidenced by the increased life expectancy since the 1940s. Many of these secondary metabolites function as antibiotics, chemotherapeutic agents, anti-fungal drugs, and/or herbicides. Indeed, the "mining" of unusual organisms such as those that grow at high temperatures, in sewers, or in interior jungles, for example, has led to a dazzling array of important therapeutic compounds such as cephalosporin, vancomycin, and erythromycin A, among others. Some of these natural products, including the antibiotics vancomycin and erythromycin A or the antitumor agent doxorubicin, contain complicated aglycone platforms, which are decorated by a variety of unusual 2,6-dideoxyhexoses and/or 2,3(4),6-trideoxyhexoses. Importantly, these sugars have been shown to provide or enhance the pharmacological properties of the macromolecules to which they are attached. The focus of this application was on the enzymes involved in the production of the unusual sugars, D-tetronitrose and D-anthrose. During the three year funding period, we determined the structures and catalytic mechanisms of four different enzymes involved in the production of these carbohydrates. The research resulted in five published papers. In addition to these basic research projects, we established a transformative outreach program for middle school students referred to as Project CRYSTAL (www.projectcrystal.org/ www.projectcrystal.org/). Project CRYSTAL had two major missions: (1) to instill a love for chemistry in middle school students by studying the inner workings of nutrition, thereby leading to healthy life choices and (2) to provide a hands-on laboratory experience in an active, state-of-the-art research laboratory, thus fostering interest in a scientific career. To achieve the first mission, we developed teaching modules based upon the chemistry of sugars, fats, and proteins. These modules included the concepts of matter, electrons, neutrons, protons, atoms, molecules, and chemical bonding. To achieve the second goal, four middle school students came to the Holden laboratory once a week during each school year to participate in academic research. These students learned fundamental techniques such as molecular cloning, protein purification, crystallization, X-ray data collection, and protein model building. Two students were co-authors on a paper published in the journal Biochemistry, and their crystals were featured on its cover.
