Yu Chi Li, Louie Tai Man, Summers Ryan, Kale Yogesh, Gopishetty Sridhar, Subramanian Mani
Center for Biocatalysis & Bioprocessing, University of Iowa, Iowa City, Iowa 52242, USA.
J Bacteriol. 2009 Jul;191(14):4624-32. doi: 10.1128/JB.00409-09. Epub 2009 May 15.
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.
恶臭假单胞菌CBB5是通过在咖啡因上富集从土壤中分离得到的。该菌株不仅能利用咖啡因、可可碱、对甲基黄嘌呤和7-甲基黄嘌呤作为唯一的碳源和氮源，还能利用茶碱和3-甲基黄嘌呤。对用过的培养基和静息细胞悬液中的代谢产物进行分析证实，CBB5最初通过一种迄今未报道的途径将茶碱N-去甲基化生成1-甲基黄嘌呤和3-甲基黄嘌呤。在以茶碱培养的CBB5的粗细胞提取物中也检测到了依赖NAD(P)H将茶碱转化为1-甲基黄嘌呤和3-甲基黄嘌呤的过程。1-甲基黄嘌呤和3-甲基黄嘌呤随后被N-去甲基化生成黄嘌呤。CBB5还分别将茶碱、1-甲基黄嘌呤和3-甲基黄嘌呤氧化为1,3-二甲基尿酸、1-甲基尿酸和3-甲基尿酸。然而，这些甲基尿酸没有进一步代谢。一种底物范围广泛的黄嘌呤氧化酶负责这些甲基尿酸的形成。相比之下，CBB5将咖啡因代谢为可可碱（主要代谢产物）和对甲基黄嘌呤（次要代谢产物）。这些二甲基黄嘌呤通过7-甲基黄嘌呤进一步N-去甲基化生成黄嘌呤。以可可碱、对甲基黄嘌呤和7-甲基黄嘌呤培养的细胞也通过相同的途径代谢上述所有甲基黄嘌呤。因此，茶碱和咖啡因的N-去甲基化途径通过不同的甲基黄嘌呤中间体在黄嘌呤处汇合。黄嘌呤最终被氧化为尿酸。当CBB5在茶碱、咖啡因或其代谢产物上生长时，参与茶碱和咖啡因降解的酶会共同表达。然而，以3-甲基黄嘌呤培养的CBB5细胞不能代谢咖啡因，而茶碱仅以较低水平代谢为甲基尿酸。据我们所知，这是关于细菌中茶碱N-去甲基化以及咖啡因和茶碱降解不同途径共同表达的首次报道。
