Public Title Engineered Deregulation of Glutamine PRPP Amidotransferase, the Key Regulatory Enzyme of Purine Biosynthesis Division Lead Inventor Zalkin, Howard Public Description Refer to Consulting Agreement Initiated by the Inventor for Licensing Information. Purine nucleotide production in bacteria is controlled and limited by gene regulation and enzyme regulation. This regulation limits the production of inosine, adenine and guanine nucleotides. Commercial production of the nucleotides is carried out using Bacillus species that are genetically deregulated. Nucleotide overproduction in these strains should be less than maximal due to feedback inhibition of glutamine PRPP amidotransferase, the regulatory enzyme of the pathway, by nucleotide endproducts. If nucleotide endproduct inhibition were decreased or abolished, purine nucleotide production should be increased. We have carried out basic studies to determine how the nucleotide endproducts bind to Bacillus subtilis glutamine PRPP amidotransferase and how inhibition takes place. This has involved an x-ray structure determination of the enzyme with bound nucleotides. Key interactions between the protein and bound nucleotides were identifed. Based on this information site-directed mutagenesis was used to construct several different deregulated enzyme mutants. The enzyme in these mutants was shown to have normal catalytic function in vivo and in vitro but to be less sensitive to nucleotide inhibition. It is proposed that individual mutations could be combined to give a multiply mutant glutamine PRPP amidotransferase having little or no sensitivity to inhibition by nucleotides. Mutant genes with single or multiple mutations can be used to replace the native gene in the commercial Bacillus strain to increase purine nucleotide production. Patent Status Public References Key Words Manager Karen White Email kfwhite@prf.org Telephone 765-588-3486 Fax 765-463-3486   Back to List