(641b) Biocatalytic Production of the High-Value Biochemical Paraxanthine from Caffeine

Paraxanthine (1,7-dimethylxanthine) is a natural product produced during metabolism of caffeine (1,3,7-trimethylxanthine) in humans and animals. Although paraxanthine is the major caffeine metabolite, its retail value is roughly 30,000 times higher than caffeine because there are limited natural sources from which to isolate the compound, and the chemical synthesis is cumbersome, low yielding, and environmentally unfavorable. Paraxanthine has multiple potential applications across various industries (i.e. pharmaceuticals, cosmetics, and food), but these applications have not been fully explored due to the high cost.

In bacteria, caffeine degradation occurs via sequential N-demethylation to xanthine. The soil bacterium Pseudomonas putida CBB5 metabolizes caffeine to xanthine using three positional-specific N-demethylase enzymes. First the N₁-demethylase, NdmA, converts caffeine to theobromine (3,7-dimethylxanthine). Theobromine is further N₃-demethylated by NdmB, resulting in 7-methylxanthine, which then undergoes N₇-demethylation by NdmC to yield xanthine. Although NdmB is active toward the N₃-methyl group, its activity is blocked by the N₁-methyl group present on the caffeine molecule. However, NdmA exhibits a slight promiscuity toward the N₃-methyl group, resulting in small amounts of paraxanthine produced from caffeine.

Recent elucidation of the NdmA and NdmB crystal structures revealed nine key amino acids involved in substrate binding. Notably, only two of the nine amino acids differ between NdmA and NdmB. Mutation of the two unique amino acids in NdmA to mimic the NdmB active site produced a mutant enzyme with a paraxanthine:theobromine ratio of at least 3:1, over a 100-fold improvement from the wild-type ratio (1:39). However, the activity was also reduced, with less than 20% of the caffeine consumed over 2 hours by E. coli expressing the mutant enzyme. Additionally, a peptide loop near the active sites also differs between NdmA and NdmB. Mutation of the NdmA loop sequence to match that of the NdmB loop increased the yield of paraxanthine to over 40%. This research confirms that biocatalytic production of paraxanthine from caffeine is achievable