Novel, highly specific N-demethylases enable bacteria to live on caffeine and related purine alkaloids.
The molecular basis for the ability of bacteria to live on caffeine as a sole carbon and nitrogen source is unknown. Pseudomonas putida CBB5, which grows on several purine alkaloids, metabolizes caffeine and related methylxanthines via sequential N-demethylation to xanthine. Metabolism of caffeine by CBB5 was previously attributed to one broad-specificity methylxanthine N-demethylase composed of two subunits, NdmA and NdmB. Here, we report that NdmA and NdmB are actually two independent Rieske nonheme iron monooxygenases with N(1)- and N(3)-specific N-demethylation activity, respectively. Activity for both enzymes is dependent on electron transfer from NADH via a redox-center-dense Rieske reductase, NdmD. NdmD itself is a novel protein with one Rieske [2Fe-2S] cluster, one plant-type [2Fe-2S] cluster, and one flavin mononucleotide (FMN) per enzyme. All ndm genes are located in a 13.2-kb genomic DNA fragment which also contained a formaldehyde dehydrogenase. ndmA, ndmB, and ndmD were cloned as His(6) fusion genes, expressed in Escherichia coli, and purified using a Ni-NTA column. NdmA-His(6) plus His(6)-NdmD catalyzed N(1)-demethylation of caffeine, theophylline, paraxanthine, and 1-methylxanthine to theobromine, 3-methylxanthine, 7-methylxanthine, and xanthine, respectively. NdmB-His(6) plus His(6)-NdmD catalyzed N(3)-demethylation of theobromine, 3-methylxanthine, caffeine, and theophylline to 7-methylxanthine, xanthine, paraxanthine, and 1-methylxanthine, respectively. One formaldehyde was produced from each methyl group removed. Activity of an N(7)-specific N-demethylase, NdmC, has been confirmed biochemically. This is the first report of bacterial N-demethylase genes that enable bacteria to live on caffeine. These genes represent a new class of Rieske oxygenases and have the potential to produce biofuels, animal feed, and pharmaceuticals from coffee and tea waste.
Department of Chemical and Biochemical Engineering, University of Iowa, Iowa City, Iowa, USA.
Ryan M
RM
Summers
Tai Man
TM
Louie
Chi-Li
CL
Yu
Lokesh
L
Gakhar
Kailin C
KC
Louie
Mani
M
Subramanian
D000470:Alkaloids / Q000737:chemistry / Q000378:metabolism*; D002110:Caffeine / Q000378:metabolism*; D020134:Catalytic Domain; D003001:Cloning, Molecular; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010089:Oxidoreductases, N-Demethylating / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D016958:Pseudomonas putida / Q000201:enzymology*; D011687:Purines / Q000737:chemistry / Q000378:metabolism*
D000470:Alkaloids; D011687:Purines; D002110:Caffeine; D010089:Oxidoreductases, N-Demethylating