RUI: Regulation of Diverse Bacterial ADP-Glucose Pyrophsophorylases

Csu Fullerton Auxiliary Services Corporation, Fullerton CA

Investigators

Abstract

There is an increasing demand for the use of the biodegradable and renewable carbon source starch in a variety of industries. The rate-limiting enzyme in the bacterial glycogen and plant starch biosynthesis pathways is ADPGlucose Pyrophosphorylase (ADPG PPase, glgC gene product) which is regulated by the binding of various allosteric effector molecules depending on the carbon utilization pathway of the organism. A complete molecular comparison of this enzyme family will allow us to perform rational engineering. The successful engineering of ADPG PPase would allow for the overproduction of starch in transgenic plants. Further, increased starch synthesis in transgenic plants could increase photosynthesis (and biomass) by decreasing feedback inhibition by organic phosphates. This project is focused on kinetic, physical, and molecular studies of the uniquely regulated bacterial ADPG PPases from a variety of sources including Rhodobacter sphaeroides, Rhodospirillum rubrum, Rhodobacter capsulatus, Rhodopseudomonas palustris, Deinococcus radiodurans, and Chlamydia trachomatis. The specific aims of this research project include I: Cloning and expression of novel glgC genes and characterization of the recombinant enzymes; and II: Protein engineering of the bacterial ADPG PPases utilizing the techniques of site-directed, truncation, chimera, and random mutagenesis well as DNA shuffling. The comprehensive approach in this project will be guided by alignment studies, a recently solved structure, molecular modeling and in silico ligand docking. Characterization of the recombinant ADPG PPases will include the measurement of binding of ligands using affinity capillary electrophoresis to complement steady-state kinetic assay data. A longer term goal includes transforming genes coding for highly active bacterial ADPG PPases into Arabidopsis thaliana leaves to determine the effects on starch accumulation, photosynthesis, and biomass. This project is well suited to training students at a primarily undergraduate institution in the theory and practice of biochemistry, analytical chemistry, molecular biology, bioinformatics, and molecular modeling within the framework of a larger interdisciplinary biotechnology project. Some of the research experiments and findings will be incorporated into lecture and lab class activities. Participating students represent three laboratories at two different California State University campuses as well as summer research students from both high schools and local community colleges. The background and experience that is gained by students in this project will open attractive opportunities in both academic and industrial careers.