Enzymes Required for the Biosynthesis of Deoxysugars Decorating Aglycone Scaffolds

University Of Wisconsin-Madison, Madison WI

Investigators

Abstract

Intellectual Merit: 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.