Journal article
Novel, Highly Specific N-Demethylases Enable Bacteria To Live on Caffeine and Related Purine Alkaloids
Journal of bacteriology, Vol.194(8), pp.2041-2049
04/01/2012
DOI: 10.1128/JB.06637-11
PMCID: PMC3318484
PMID: 22328667
Abstract
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.
Details
- Title: Subtitle
- Novel, Highly Specific N-Demethylases Enable Bacteria To Live on Caffeine and Related Purine Alkaloids
- Creators
- Ryan M. Summers - University of IowaTai Man Louie - University of IowaChi-Li Yu - Univ Iowa, Dept Chem & Biochem Engn, Iowa City, IA 52242 USALokesh Gakhar - University of IowaKailin C. Louie - University of IowaMani Subramanian - University of Iowa
- Resource Type
- Journal article
- Publication Details
- Journal of bacteriology, Vol.194(8), pp.2041-2049
- Publisher
- Amer Soc Microbiology
- DOI
- 10.1128/JB.06637-11
- PMID
- 22328667
- PMCID
- PMC3318484
- ISSN
- 0021-9193
- eISSN
- 1098-5530
- Number of pages
- 9
- Grant note
- 0821615 / Division Of Earth Sciences; Directorate For Geosciences; National Science Foundation (NSF); NSF - Directorate for Geosciences (GEO) University of Iowa
- Language
- English
- Date published
- 04/01/2012
- Academic Unit
- Medicine Administration
- Record Identifier
- 9984622753502771
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