Catabolism of l-phenylalanine and l-tyrosine by Rhodobacter sphaeroides OU5 occurs through 3,4-dihydroxyphenylalanine

Rhodobacter sphaeroides OU5 utilized l-phenylalanine as sole source of nitrogen for growth. The metabolites of l-phenylalanine catabolism, i.e. 4-hydroxy phenylalanine (l-tyrosine), 3,4-dihydroxyphenylalanine (DOPA), 3,4-dihydroxyphenyl-pyruvic acid (DOPP), 3,4-dihydroxyphenyllactic acid (DOPLA), 3,4-dihydroxyphenyl-acetic acid (DOPAc) and 3,4-dihydroxybenzoic acid (PC), were identified using liquid chromatography-mass spectroscopy (LC-MS). With 2-oxoglutarate as an amino acceptor, DOPA aminotransferase activity was observed with cell-free extracts and the product DOPP was confirmed through mass analysis. Reductive deamination of DOPA also occurred in the absence of 2-oxoglutarate, whose products were 3,4-dihydroxyphenylpropionic acid (DPPA) and ammonia. The enzyme DOPA-reductive deaminase (DOPARDA) was purified to its homogeneity and characterized. DOPARDA has an obligate requirement for NADH and is functional at low concentrations of the substrate (<150 μM). The molecular mass of the purified enzyme was ≈274 kD and the enzyme could be a heterotetramer of 110, 82, 43 and 39 kD subunits as determined by SDS-PAGE.