title: Delineation of the caffeine C-8 oxidation pathway in Pseudomonas sp. strain CBB1 via characterization of a new trimethyluric acid monooxygenase and genes involved in trimethyluric acid metabolism.
abstract: The molecular basis of the ability of bacteria to live on caffeine via the C-8 oxidation pathway is unknown. The first step of this pathway, caffeine to trimethyluric acid (TMU), has been attributed to poorly characterized caffeine oxidases and a novel quinone-dependent caffeine dehydrogenase. Here, we report the detailed characterization of the second enzyme, a novel NADH-dependent trimethyluric acid monooxygenase (TmuM), a flavoprotein that catalyzes the conversion of TMU to 1,3,7-trimethyl-5-hydroxyisourate (TM-HIU). This product spontaneously decomposes to racemic 3,6,8-trimethylallantoin (TMA). TmuM prefers trimethyluric acids and, to a lesser extent, dimethyluric acids as substrates, but it exhibits no activity on uric acid. Homology models of TmuM against uric acid oxidase HpxO (which catalyzes uric acid to 5-hydroxyisourate) reveal a much bigger and hydrophobic cavity to accommodate the larger substrates. Genes involved in the caffeine C-8 oxidation pathway are located in a 25.2-kb genomic DNA fragment of CBB1, including cdhABC (coding for caffeine dehydrogenase) and tmuM (coding for TmuM). Comparison of this gene cluster to the uric acid-metabolizing gene cluster and pathway of Klebsiella pneumoniae revealed two major open reading frames coding for the conversion of TM-HIU to S-(+)-trimethylallantoin [S-(+)-TMA]. The first one, designated tmuH, codes for a putative TM-HIU hydrolase, which catalyzes the conversion of TM-HIU to 3,6,8-trimethyl-2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (TM-OHCU). The second one, designated tmuD, codes for a putative TM-OHCU decarboxylase which catalyzes the conversion of TM-OHCU to S-(+)-TMA. Based on a combination of enzymology and gene-analysis, a new degradative pathway for caffeine has been proposed via TMU, TM-HIU, TM-OHCU to S-(+)-TMA.
authors.affiliation: Department of Chemical and Biochemical Engineering, University of Iowa, Iowa City, Iowa, USA.
authors.forename: Sujit Kumar
authors.initials: SK
authors.lastname: Mohanty
authors.forename: Chi-Li
authors.initials: CL
authors.lastname: Yu
authors.forename: Shuvendu
authors.initials: S
authors.lastname: Das
authors.forename: Tai Man
authors.initials: TM
authors.lastname: Louie
authors.forename: Lokesh
authors.initials: L
authors.lastname: Gakhar
authors.forename: Mani
authors.initials: M
authors.lastname: Subramanian
mesh_terms: D002110:Caffeine / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D007711:Klebsiella pneumoniae / Q000201:enzymology / Q000235:genetics; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D005810:Multigene Family; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D017422:Sequence Analysis, DNA; D026801:Synteny; D014527:Uric Acid / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D004269:DNA, Bacterial; D014527:Uric Acid; D002110:Caffeine; C039222:1,3,7-trimethyluric acid; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases