Publications

• Enzymological aspects of caffeine demethylation and formaldehyde oxidation by Pseudomonas putida C1.

  Hohnloser W., Osswald B., Lingens F.

  1) The enzymatic demethylation of caffeine (1,3,7-trimethylxanthine) by Pseudomonas putida C1 was investigated; an inducible enzyme system has been observed. This enzyme shows an optimum pH of about 6.0, and the optimum temperature is in the range of 22-24 degrees C. The enzyme is absolutely depen ... >> More

  1) The enzymatic demethylation of caffeine (1,3,7-trimethylxanthine) by Pseudomonas putida C1 was investigated; an inducible enzyme system has been observed. This enzyme shows an optimum pH of about 6.0, and the optimum temperature is in the range of 22-24 degrees C. The enzyme is absolutely dependent on NADH or NADPH as a cosubstrate and is activated by CO2+. 2) The formaldehyde generated by the demethylation of caffeine is oxidized by an NAD-dependent formaldehyde dehydrogenase, which is independent of Mg2+ and glutathione. The enzyme was purified from cell-free extracts of Pseudomonas putida C1 by DEAE-cellulose, Sephadex G-150 and Sephadex A-50 chromatography. The purified enzyme was homogeneous as judged by polyacrylamide gel electrophoresis and was most active at a pH between 8.5 and 9.0. The molecular weight was estimated to be about 250,000 by the gel filtration method. Kinetic analysis gave KM values of about 0.2 mM for formaldehyde and 0.5 mM for NAD+. << Less

  Hoppe Seylers Z Physiol Chem 361:1763-1766(1980) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Novel, highly specific N-demethylases enable bacteria to live on caffeine and related purine alkaloids.

  Summers R.M., Louie T.M., Yu C.L., Gakhar L., Louie K.C., Subramanian M.

  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 ... >> More

  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. << Less

  J. Bacteriol. 194:2041-2049(2012) [PubMed] [EuropePMC]

  This publication is cited by 11 other entries.

• Characterization of a broad-specificity non-haem iron N-demethylase from Pseudomonas putida CBB5 capable of utilizing several purine alkaloids as sole carbon and nitrogen source.

  Summers R.M., Louie T.M., Yu C.L., Subramanian M.

  N-Demethylation of many xenobiotics and naturally occurring purine alkaloids such as caffeine and theobromine is primarily catalysed in higher organisms, ranging from fungi to mammals, by the well-studied membrane-associated cytochrome P450s. In contrast, there is no well-characterized enzyme for ... >> More

  N-Demethylation of many xenobiotics and naturally occurring purine alkaloids such as caffeine and theobromine is primarily catalysed in higher organisms, ranging from fungi to mammals, by the well-studied membrane-associated cytochrome P450s. In contrast, there is no well-characterized enzyme for N-demethylation of purine alkaloids from bacteria, despite several reports on their utilization as sole source of carbon and nitrogen. Here, we provide what we believe to be the first detailed characterization of a purified N-demethylase from Pseudomonas putida CBB5. The soluble N-demethylase holoenzyme is composed of two components, a reductase component with cytochrome c reductase activity (Ccr) and a two-subunit N-demethylase component (Ndm). Ndm, with a native molecular mass of 240 kDa, is composed of NdmA (40&emsp14;kDa) and NdmB (35&emsp14;kDa). Ccr transfers reducing equivalents from NAD(P)H to Ndm, which catalyses an oxygen-dependent N-demethylation of methylxanthines to xanthine, formaldehyde and water. Paraxanthine and 7-methylxanthine were determined to be the best substrates, with apparent K(m) and k(cat) values of 50.4±6.8&emsp14;μM and 16.2±0.6&emsp14;min(-1), and 63.8±7.5&emsp14;μM and 94.8±3.0&emsp14;min(-1), respectively. Ndm also displayed activity towards caffeine, theobromine, theophylline and 3-methylxanthine, all of which are growth substrates for this organism. Ndm was deduced to be a Rieske [2Fe-2S]-domain-containing non-haem iron oxygenase based on (i) its distinct absorption spectrum and (ii) significant identity of the N-terminal sequences of NdmA and NdmB with the gene product of an uncharacterized caffeine demethylase in P. putida IF-3 and a hypothetical protein in Janthinobacterium sp. Marseille, both predicted to be Rieske non-haem iron oxygenases. << Less

  Microbiology 157:583-592(2011) [PubMed] [EuropePMC]

  This publication is cited by 11 other entries.