This research is focused on kinetic, physical, molecular, and metabolic studies of the uniquely regulated bacterial ADPG Ppases, the rate-limiting enzymes in the glycogen and starch biosynthetic pathways, from the following sources: Rhodobacter sphaeroides, Rhodospirillum rubrum, Agrobacterium tumefaciens, and Rhodobacter capsulatus. The activity of ADPG Ppase is modulated by the binding of various allosteric effector molecules depending on the carbon utilization pathway of the organism. A complete molecular comparison of this family of enzymes will allow us to perform rational protein engineering with the goal of enhancing function. The successful engineering of ADPG Ppase would allow for the overproduction of starch in transgenic plants. The specific aims of this research project are I: Identification of the amino acids important for function and regulation and physical characterization of these ADPG Ppases; II: Cloning and sequencing of unique bacterial ADPG Ppase genes; III: Expression and protein engineering of ADPG Ppases; and, longer term, IV: Elucidating the effect of variant ADPG Ppases and glycogen/starch metabolism enzymes on the structure of the end-product starch. This interdisciplinary approach will allow for a detailed picture of structure/function relationships to emerge. Information for rational mutagenesis (Aim III.) will be derived in part from the results of Aims I and II. The techniques utilized in order to identify and characterize the amino acids in the various active and allosteric activator and inhibitor site(s) include enzyme kinetics, chemical modification, limited proteolysis, X-ray crystallography, cloning and sequencing of genes (including use of PCR), protein expression, bioinformatics (alignment of nucleic acid and protein sequences), and site-directed and random mutagenesis. Long-range goals include a comparison between mutant and native crystal structures in parallel with kinetic and other functional analyses of the mutants. Experiments in development will also involve utilizing engineered ADPG Ppases in both in vivo and in vitro recombinant systems in combination with various starch synthases, branching, and debranching enzymes to elucidate structure/function relationships of the end product starch. The glucan produced from these systems will be isolated and analyzed with respect to yield, chain length, and branching pattern. The regulation of the glycogen and starch biosynthetic pathways is a growing area of interest due to the increasing demand for natural and modified starches in a variety of industries. These renewable and biodegradable carbon sources can serve as inexpensive starting materials for bio-ethanol, organic acids, and antibiotic synthesis and have great potential for use in the making of specialty plastics, adhesives, detergents, surfactants, and packaging materials. Beyond contributing to enzymology and agricultural biotechnology, this project is well suited to training students at a primarily undergraduate institution in the theory and practice of biochemistry, molecular biology, and biotechnology. The background and experience students gain in the laboratory makes them attractive candidates for both academic and industrial positions.
