Two Distinct Pathways for Metabolism of Theophylline and Caffeine Are Coexpressed in Pseudomonas putida CBB5▿ †

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

MOESM1 of Direct conversion of theophylline to 3-methylxanthine by metabolically engineered E. coli

Additional file 1: Table S1. Prices of various natural methylxanthines obtained from the Sigma–Aldrich website on 18 September, 2015. Table S2. Estimated copy number of ndmA and ndmD genes in strains used in this study. Table S3. Comparison of growth and activity of resting cell suspensions of strains pDdA, pDdAA, and pAD1dDD. Concentrations of TP, 3MX, and 1MX after 90 min are reported as means with standard deviations of triplicate reactions. Table S4. Comparison of growth and activity of resting cell suspension of strain pDdA grown in LB and SB. Concentrations of TP, 3MX, and 1MX after 2 h are reported as means with standard deviations of triplicate reactions. Figure S1. Maps of plasmids in strains used to produce 3MX from TP. ori_pBR322, pBR322 origin of replication; ori_P15A, P15A origin of replication; Amp-R, ampicillin resistance gene, Kan-R, kanamycin resistance gene, CAT, chloramphenicol resistance gene, ndmA, N1-demethylase gene; ndmD, N-demethylase reductase gene; His-ndmD, N-terminal His6-tagged N-demethylase reductase gene; T7, T7 promoter. Plasmids beginning with “p” use pET backbones, plasmids beginning with “d” use the pACYCDuet-1 plasmid backbone. Figure S2. SDS-PAGE analysis of ndmA and ndmD expression in metabolically engineered strains of E. coli. A total of 10 μg protein was loaded into each well. Molecular weights of markers (in kDa) are shown to the left of the gel. Blue arrows indicate NdmA and NdmD protein bands. Lane 1, pAD1 soluble fraction; lane 2, pAD1 insoluble fraction; lane 3, pDdA soluble fraction; lane 4, pDdA insoluble fraction; lane 5, BL21(DE3) soluble fraction (negative control); lane 6, molecular weight standard; lane 7, pAD1DD soluble fraction; lane 8, pAD1DD insoluble fraction; lane 9, pDdAA soluble fraction; lane 10, pDdAA insoluble fraction. Figure S3. Separation of 3MX and 1MX by preparative chromatography. Retention times of 3MX and 1MX are 116 and 135 min, respectively. Figure S4. NMR of 3-methylxanthine. (A) NMR of 3MX standard obtained from Sigma Aldrich. (B) NMR of biologically produced and purified 3MX sample produced in this wor

A Novel Caffeine Dehydrogenase in Pseudomonas sp. Strain CBB1 Oxidizes Caffeine to Trimethyluric Acid▿

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 Q0 was the preferred electron acceptor. The enzyme was specific for caffeine and theobromine and showed no activity with xanthine