{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,9]],"date-time":"2026-05-09T21:11:25Z","timestamp":1778361085883,"version":"3.51.4"},"reference-count":55,"publisher":"Microbiology Society","issue":"2","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2011,2,1]]},"abstract":"<jats:p><jats:italic>N<\/jats:italic>-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<jats:italic>N<\/jats:italic>-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<jats:italic>N<\/jats:italic>-demethylase from<jats:italic>Pseudomonas putida<\/jats:italic>CBB5. The soluble<jats:italic>N<\/jats:italic>-demethylase holoenzyme is composed of two components, a reductase component with cytochrome<jats:italic>c<\/jats:italic>reductase activity (Ccr) and a two-subunit<jats:italic>N<\/jats:italic>-demethylase component (Ndm). Ndm, with a native molecular mass of 240\u200akDa, is composed of NdmA (40\u2005kDa) and NdmB (35\u2005kDa). Ccr transfers reducing equivalents from NAD(P)H to Ndm, which catalyses an oxygen-dependent<jats:italic>N<\/jats:italic>-demethylation of methylxanthines to xanthine, formaldehyde and water. Paraxanthine and 7-methylxanthine were determined to be the best substrates, with apparent<jats:italic>K<\/jats:italic><jats:sub>m<\/jats:sub>and<jats:italic>k<jats:sub>cat<\/jats:sub><\/jats:italic>values of 50.4\u00b16.8\u2005\u03bcM and 16.2\u00b10.6\u2005min<jats:sup>\u22121<\/jats:sup>, and 63.8\u00b17.5\u2005\u03bcM and 94.8\u00b13.0\u2005min<jats:sup>\u22121<\/jats:sup>, 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\u20132S]-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<jats:italic>P. putida<\/jats:italic>IF-3 and a hypothetical protein in<jats:italic>Janthinobacterium<\/jats:italic>sp. Marseille, both predicted to be Rieske non-haem iron oxygenases.<\/jats:p>","DOI":"10.1099\/mic.0.043612-0","type":"journal-article","created":{"date-parts":[[2010,10,22]],"date-time":"2010-10-22T04:45:17Z","timestamp":1287722717000},"page":"583-592","source":"Crossref","is-referenced-by-count":52,"title":["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"],"prefix":"10.1099","volume":"157","author":[{"given":"Ryan M.","family":"Summers","sequence":"first","affiliation":[{"name":"Department of Chemical and Biochemical Engineering and Center for Biocatalysis and Bioprocessing, University of Iowa, Iowa City, IA 52242, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tai Man","family":"Louie","sequence":"additional","affiliation":[{"name":"Department of Chemical and Biochemical Engineering and Center for Biocatalysis and Bioprocessing, University of Iowa, Iowa City, IA 52242, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chi Li","family":"Yu","sequence":"additional","affiliation":[{"name":"Department of Chemical and Biochemical Engineering and Center for Biocatalysis and Bioprocessing, University of Iowa, Iowa City, IA 52242, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mani","family":"Subramanian","sequence":"additional","affiliation":[{"name":"Department of Chemical and Biochemical Engineering and Center for Biocatalysis and Bioprocessing, University of Iowa, Iowa City, IA 52242, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"345","reference":[{"key":"R1","doi-asserted-by":"crossref","first-page":"743","DOI":"10.1016\/S0141-0229(03)00207-2","article-title":"The synthesis of buprenorphine intermediates by regioselective microbial N - 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Isolation of homogenous reduced diphosphopyridine nucleotide-rubredoxin reductase","volume":"247","author":"Ueda","year":"1972","journal-title":"J Biol Chem"},{"key":"R51","doi-asserted-by":"crossref","first-page":"229","DOI":"10.1146\/annurev.nu.02.070182.001305","article-title":"Cellular folate binding proteins; function and significance","volume":"2","author":"Wagner","year":"1982","journal-title":"Annu Rev Nutr"},{"key":"R52","doi-asserted-by":"crossref","first-page":"1088","DOI":"10.1128\/JB.123.3.1088-1106.1975","article-title":"Metabolism of N -methylpurines by a Pseudomonas putida strain isolated by enrichment on the caffeine as sole source of carbon and nitrogen","volume":"123","author":"Woolfolk","year":"1975","journal-title":"J Bacteriol"},{"key":"R53","doi-asserted-by":"crossref","first-page":"311","DOI":"10.1007\/s10295-006-0199-8","article-title":"Purification, characterization, and crystallization of the components of a biphenyl dioxygenase system from Sphingobium yanoikuyae B1","volume":"34","author":"Yu","year":"2007","journal-title":"J Ind Microbiol Biotechnol"},{"key":"R54","doi-asserted-by":"crossref","first-page":"772","DOI":"10.1128\/JB.01390-07","article-title":"A novel caffeine dehydrogenase in Pseudomonas sp. strain CBB1 oxidizes caffeine to trimethyluric acid","volume":"190","author":"Yu","year":"2008","journal-title":"J Bacteriol"},{"key":"R55","doi-asserted-by":"crossref","first-page":"4624","DOI":"10.1128\/JB.00409-09","article-title":"Two distinct pathways for metabolism of theophylline and caffeine are coexpressed in Pseudomonas putida CBB5","volume":"191","author":"Yu","year":"2009","journal-title":"J Bacteriol"}],"container-title":["Microbiology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.microbiologyresearch.org\/content\/journal\/micro\/10.1099\/mic.0.043612-0?crawler=true","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,11,12]],"date-time":"2021-11-12T12:23:44Z","timestamp":1636719824000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.microbiologyresearch.org\/content\/journal\/micro\/10.1099\/mic.0.043612-0"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2011,2,1]]},"references-count":55,"journal-issue":{"issue":"2","published-print":{"date-parts":[[2011,2,1]]}},"URL":"https:\/\/doi.org\/10.1099\/mic.0.043612-0","relation":{},"ISSN":["1350-0872","1465-2080"],"issn-type":[{"value":"1350-0872","type":"print"},{"value":"1465-2080","type":"electronic"}],"subject":[],"published":{"date-parts":[[2011,2,1]]}}}