%0 Journal Article %A Shelby Brooks Mills %A Meredith B. Mock %A Ryan M. Summers %T Rational Protein Engineering of Bacterial N-demethylases to Create Biocatalysts for the Production of Methylxanthines %D 2021 %R 10.1101/2021.12.17.472166 %J bioRxiv %P 2021.12.17.472166 %X Methylxanthines have a rich history as therapeutics and pharmaceuticals. However, natural dimethyl- and monomethylxanthines are difficult to produce synthetically, which has limited further exploration of these compounds in medicinal applications. A biosynthetic method for production of methylxanthines from whole cell biocatalysts is an attractive alternative. The bacterium Pseudomonas putida CBB5 contains a set of five enzymes, NdmABCDE, which are responsible for methylxanthine metabolism via N-demethylation to xanthine. The recent elucidation of the crystal structures of NdmA and NdmB, which remove the N1- and N3-methyl groups of caffeine, respectively, has opened new avenues to create biocatalysts for methylxanthine production. We have created a set of fifteen N-demethylase mutants and expressed them in E. coli BL21(DE3) as whole cell biocatalysts. The activity of each mutant was characterized for their affinity towards caffeine, theobromine, and theophylline. Two mutant enzymes in particular, labeled NdmA3 and NdmA4, both exhibited selectivity towards the N3-methyl group instead of the N1-methyl group. We also discovered that specific point mutations in NdmD resulted in the ability to tune the rate of the N-demethylase reaction. These new enzymes provide the capability of producing high-value methylxanthines, such as paraxanthine and 1-methylxanthine, through a biocatalytic route.Competing Interest StatementThe authors have declared no competing interest. %U https://www.biorxiv.org/content/biorxiv/early/2021/12/18/2021.12.17.472166.full.pdf