Cloning and expression of the novel glgC genes from Chlamydia trachomatis and Bacillus subtilis and directed evolution of the recombinant Chlamydia ADP-glucose pyrophosphorylase

ADP-Glucose pyrophosphorylase (ADPGlc PPase, glgC gene product, EC 2.7.7.27) is the rate limiting step in glycogen biosynthesis and catalyzes the conversion of glucose-1-phosphate and adenosine triphosphate to ADP-glucose and pyrophosphate. With the influx of data from genome projects, it is of interest to clone, express, and characterize diverse ADPGlc PPase further probing structure/function relationships. We have successfully cloned and expressed ADPGlc PPases from the following bacteria: Bacillus subtilis (a two subunit system) and Chlamydia trachomatis . Alignment studies with these novel ADPGlc PPases revealed differences in primary amino acid sequence in key regions from previously characterized ADPGlc PPases.; To further elucidate structure function relationships the ADPGlc PPase from C. trachomatis was subjected to directed molecular evolution by DNA shuffling and random mutagenesis. The resulting mutant enzymes had increases in specific activity 40 fold higher than wild type and identified key amino acid residues in the primary gene sequence.