Sugar-containing compounds, also called carbohydrates, are a broad class of chemicals beyond the sucrose that makes sweets sweet. Carbohydrates are important to recognition processes in biology because of the diversity of their structures. Each structure can be equated with a distinct message when it is bound by the appropriate protein partner. Over the past 15 years, our group has developed ways to make molecules that mimic some of the properties of natural carbohydrates but are also wholly un-natural. These carbohydrate mimics are characterized by their ring size: most natural carbohydrates are composed of six-atom ring units (pyranoses) but ours are made of seven atoms (septanoses). We have also investigated how these “ring-expanded” sugars bind to proteins and have developed some rules for understanding the protein-carbohydrate interactions. Over the period of the current grant, our objective was to take enzymes that usually cleave the linkages between natural sugar rings – called glycosidases - and and characterize how they bind to our ring-expanded sugars.

The intellectual merits of the project have arisen from the development of the methods to synthesize septanoses that could act as substrates of the enzymes, characterization of enzyme activity, and investigation of how the binding and catalysis occurred. Our strategy for synthesizing septanoses relies on derivatization of naturally occurring pyranose sugars and devising routes to attach another carbon into the ring. In addition, we had to establish a way to link the sugar to another molecule such that, when the key bond is broken by the enzyme, a reaction product that is easily observed by spectroscopy is generated. For screening large families of mutant enzymes (explained below), we also synthesized derivatives that would give a blue-colored compound upon cleavage by the enzyme. The synthetic routes we established during the project will enable other groups to investigate the cleavage reaction by other glycosidases.

With our collaborators in the group of Toni Planas at the Institute of Chemistry in Sarria, Barcelona, Spain, we found low enzyme activity (>10,000x slower) by the Streptomyces glycosidase we chose to investigate. We had anticipated poor catalysis and established a method to search for mutants with increased activity. Mutant libraries that varied several key residues were screened with the chromogenic septanose substrate but mutants with increased activity were not revealed. Control experiments revealed that the septanose sugar may have been binding in the wrong position on the enzyme, at a location where the penultimate sugar of the carbohydrate binds. A set of rigorous, in silico Docking experiments were therefore undertaken to provide clues to the true binding mode of the septanose in the active site of the glycosidase. These computational experiments revealed a key difference in how the enzyme binds our septanoses, and has provided design features for a subsequent round of septanose substrates.

The project has allowed us to address some fundamental aspects of protein-carbohydrate interactions through synthesis of un-natural seven membered-ring septanose sugars. We have learned about the structure and function of these carbohydrate mimics and their potential applications in glycobiology. Our interdisciplinary, international collaboration has broader impacts in the training of young chemists and protein engineers. Further, through our collaboration, students in both our group and the Planas group have had an opportunity for technical and social interactions.

Last Modified: 11/14/2019
Modified by: Mark Peczuh