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cThe 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 Ndemethylase 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. Many natural products and xenobiotic compounds contain N-linked methyl groups. A search of the Combined Chemical Dictionary database (http://ccd.chemnetbase.com) identified 19,091 compounds out of approximately 500,000 entries that contain at least one N-methyl group. N-Demethylations of many of these compounds by members of cytochrome P450, flavoenzyme, and 2-ketoglutarate-dependent nonheme iron oxygenase families are critical biological processes in living organisms (1,12,17,27,31). These processes include detoxification of drugs and xenobiotic compounds, regulation of chromatin dynamics and gene transcription, and repair of alkylation damages in purine and pyrimidine bases in nucleic acids. Members of all aforementioned enzyme families also catalyze O-demethylation reactions (14). Bacteria have evolved highly specific Rieske [2Fe-2S] domaincontaining O-demethylases that belong to the Rieske oxygenase (RO) family for the degradation of methoxybenzoates (5, 16). However, to the best of our knowledge, there is no description of N-demethylation by ROs.Caffeine (1,3,7-trimethylxanthine) and related N-methylated xanthines are purine alkaloids that are ext...

The naphthalene dioxygenase (NDO) system catalyzes the first step in the degradation of naphthalene by Pseudomonas sp. strain NCIB 9816-4. The enzyme has a broad substrate range and catalyzes several types of reactions including cis-dihydroxylation, monooxygenation, and desaturation. Substitution of valine or leucine at Phe-352 near the active site iron in the ␣ subunit of NDO altered the stereochemistry of naphthalene cis-dihydrodiol formed from naphthalene and also changed the region of oxidation of biphenyl and phenanthrene. In this study, we replaced Phe-352 with glycine, alanine, isoleucine, threonine, tryptophan, and tyrosine and determined the activity with naphthalene, biphenyl, and phenanthrene as substrates. NDO variants F352W and F352Y were marginally active with all substrates tested. F352G and F352A had reduced but significant activity, and F352I, F352T, F352V, and F352L had nearly wild-type activities with respect to naphthalene oxidation. All active enzymes had altered regioselectivity with biphenyl and phenanthrene. In addition, the F352V and F352T variants formed the opposite enantiomer of biphenyl cis-3,4-dihydrodiol [77 and 60% (؊)-(3S,4R), respectively] to that formed by wild-type NDO [>98% (؉)-(3R,4S)]. The F352V mutant enzyme also formed the opposite enantiomer of phenanthrene cis-1,2-dihydrodiol from phenanthrene to that formed by biphenyl dioxygenase from Sphingomonas yanoikuyae B8/36. A recombinant Escherichia coli strain expressing the F352V variant of NDO and the enantioselective toluene cis-dihydrodiol dehydrogenase from Pseudomonas putida F1 was used to produce enantiomerically pure (؊)-biphenyl cis-(3S,4R)-dihydrodiol and (؊)-phenanthrene cis-(1S,2R)-dihydrodiol from biphenyl and phenanthrene, respectively.The naphthalene dioxygenase (NDO) system (EC 1.14.12.12) catalyzes the first step in the degradation of naphthalene in Pseudomonas sp. NCIB 9816-4. In this reaction, both atoms of O 2 are added to the aromatic ring to form (ϩ)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene (naphthalene cis-dihydrodiol) (28,29). NDO consists of three components. An iron-sulfur flavoprotein reductase and a Rieske iron-sulfur ferredoxin transfer electrons from NAD(P)H to the catalytic oxygenase component (15,16,21,22). The oxygenase consists of large (␣) and small (␤) subunits that form an ␣ 3 ␤ 3 native structure (31). Each ␣ subunit contains a Rieske [2Fe-2S] center and mononuclear nonheme iron (15, 31). Electrons are transferred from the Rieske center in one ␣ subunit to the mononuclear iron in an adjacent ␣ subunit (31, 39), and this is the site of oxygen activation and catalysis.NDO catalyzes the oxidation of a wide variety of aromatic compounds, and many of the products are enantiomerically pure chiral compounds (9, 24, 45). The use of dioxygenases to initiate biocatalytic routes for the production of pharmaceuticals and natural products has received significant attention of late (9,12,25,42), and the possibility of generating new synthons with opposite stereochemistry is an attractive alternativ...

A unique heterotrimeric caffeine dehydrogenase was purified from Pseudomonas sp. strain CBB1. This enzyme oxidized caffeine to trimethyluric acid stoichiometrically and hydrolytically, without producing hydrogen peroxide. The enzyme was not NAD(P) ؉ dependent; coenzyme Q 0 was the preferred electron acceptor. The enzyme was specific for caffeine and theobromine and showed no activity with xanthine.

Pseudomonas putida CBB5 was isolated from soil by enrichment on caffeine. This strain used not only caffeine, theobromine, paraxanthine, and 7-methylxanthine as sole carbon and nitrogen sources but also theophylline and 3-methylxanthine. Analyses of metabolites in spent media and resting cell suspensions confirmed that CBB5 initially N demethylated theophylline via a hitherto unreported pathway to 1-and 3-methylxanthines. NAD(P)H-dependent conversion of theophylline to 1-and 3-methylxanthines was also detected in the crude cell extracts of theophylline-grown CBB5. 1-Methylxanthine and 3-methylxanthine were subsequently N demethylated to xanthine. CBB5 also oxidized theophylline and 1-and 3-methylxanthines to 1,3-dimethyluric acid and 1-and 3-methyluric acids, respectively. However, these methyluric acids were not metabolized further. A broad-substrate-range xanthine-oxidizing enzyme was responsible for the formation of these methyluric acids. In contrast, CBB5 metabolized caffeine to theobromine (major metabolite) and paraxanthine (minor metabolite). These dimethylxanthines were further N demethylated to xanthine via 7-methylxanthine. Theobromine-, paraxanthine-, and 7-methylxanthine-grown cells also metabolized all of the methylxanthines mentioned above via the same pathway. Thus, the theophylline and caffeine N-demethylation pathways converged at xanthine via different methylxanthine intermediates. Xanthine was eventually oxidized to uric acid. Enzymes involved in theophylline and caffeine degradation were coexpressed when CBB5 was grown on theophylline or on caffeine or its metabolites. However, 3-methylxanthine-grown CBB5 cells did not metabolize caffeine, whereas theophylline was metabolized at much reduced levels to only methyluric acids. To our knowledge, this is the first report of theophylline N demethylation and coexpression of distinct pathways for caffeine and theophylline degradation in bacteria.Caffeine (1,3,7-trimethylxanthine) and related methylxanthines are widely distributed in many plant species. Caffeine is also a major human dietary ingredient that can be found in common beverages and food products, such as coffee, tea, and chocolates. In pharmaceuticals, caffeine is used generally as a cardiac, neurological, and respiratory stimulant, as well as a diuretic (3). Hence, caffeine and related methylxanthines enter soil and water easily through decomposed plant materials and other means, such as effluents from coffee-and tea-processing facilities. Therefore, it is not surprising that microorganisms capable of degrading caffeine have been isolated from various natural environments, with or without enrichment procedures (3, 10). Bacteria use oxidative and N-demethylating pathways for catabolism of caffeine. Oxidation of caffeine by a Rhodococcus sp.-Klebsiella sp. mixed-culture consortium at the C-8 position to form 1,3,7-trimethyluric acid (TMU) has been reported (8). An 85-kDa, flavin-containing caffeine oxidase was purified from this consortium (9). Also, Mohapatra et al. (12) purified a 65-kDa...