Structural Investigations of Caffeine Metabolic Enzymes

Pseudomonas putida CBB5 digests caffeine and related purine alkaloids through an N-demethylation pathway. Five key enzymes have been found responsible for this: NdmA, NdmB, NdmC, NdmD, and NdmE. Classical bioengineering techniques were used to further investigate these enzymes. When a His₆-tagged NdmB and a non-His₆ tagged NdmA were co-expressed in E. coli and cell lysate passed through a Ni-NTA column, both were retained on the column and co eluted with 500 mM imidazole. These results indicate that NdmA and NdmB form an enzymatic complex when coexpressed in vivo. The ultimate goal of this project is to create chimeric enzymes combining NdmA or NdmB with NdmC. Toward this end, homology models of the enzymes were analyzed and the chimeric enzymes were designed in silico. The chimeric genes have also been cloned into E. coli expression vectors and activity assays are pending. Caffeine and its metabolites are in high demand due to their use in food, beverages, and pharmaceuticals. By understanding more about the enzymes themselves, it is expected that the reaction pathways can be better controlled for more efficient yields of valuable biochemicals.