EAPSI: Crystallization of Mutant N-demethylase Enzymes

Brooks Shelby G, Tuscaloosa AL

Investigators

Abstract

The goal of this research project is to determine the crystal structures of mutant enzymes derived from the bacterial caffeine-degrading enzymes NdmABCDE. These enzymes are used to sequentially remove the N1-, N3, and N7-methyl groups from the caffeine molecule. This N-demethylation process can result in a new, economical, and environmentally-friendly method to produce valuable methylxanthies from caffeine. NdmA and NdmB perform the first two steps by removing the N1-methyl group followed by the N3-methyl group, respectively. NdmCDE form a large protein complex to carry out the N7-demethylation of 7-methylxanthine to xanthine. We have created several mutant N¬-demethylase enzymes with improved abilities to produce high-value metabolites from caffeine, but the yields of these metabolites are still low. Crystal structures of the mutant caffeine-degrading enzymes with the metabolites they degrade will be determined under the mentorship of Dr. Hyun Kyu Song of Korea University. The crystal structures will provide insight into rationally designing new mutant enzymes to increase the yield of high-value biochemicals from this process. Dr. Song's research group is the only one to determine the structure of the N-demethylase enzymes to date. The Song lab, thus, has all of the protocols and equipment necessary to complete this project. The N-demethylation of caffeine (1,3,7-trimethylxanthine) can provide valuable methylxanthines in an economical, environmentally-friendly method compared to the current chemical synthesis. Paraxanthine (1,7-dimethylxanthine) is produced as a minor product through the N3-demethylation of caffeine due to slight promiscuity of the N1-demethylase NdmA toward the N3-methyl group. The N3-demethylase NdmB catalyzes the conversion of theobromine (3,7-dimethylxanthine) to 7-methylxanthine, but exhibits very low activity toward molecules that contain an N1-methyl group. Mutants of NdmA and NdmB have been created to better understand the substrate interactions with the enzymes. The hypothesis of this work is that the mutation of two residues in the NdmA active site to those found at similar locations in NdmB will swap the NdmA activity from N1-demethylation to N3-demethylation, resulting in higher yields of paraxanthine. Under the direction of Dr. Song, the researcher will determine the crystal structures of these mutants with their substrates and discover the substrate orientation in the enzyme to ultimately optimize the yield of paraxanthine. Additionally, the project will also seek to determine the crystal structure of the native NdmCDE complex. The knowledge gained through these crystallographic studies will enable rational engineering of bacterial N-demethylase enzymes and accelerate the biosynthesis of high-value methylxanthines for applications in the pharmaceutical and cosmetic industries. This award, under the East Asia and Pacific Summer Institutes program, supports summer research by a U.S. graduate student and is jointly funded by NSF and the National Research Foundation of Korea.