ABSTRACT
An enzyme involved in the catabolism of 3,4-dihydroxyphenylalanine (DOPA) was isolated from Rhodobacter sphaeroides OU5. The enzyme catalyzes the formation of 3,4-dihydroxyphenylpyruvic acid (DOPP) and ammonia from DOPA. Formation of ammonia by DOPA oxidative deaminase was O2 dependent and the enzyme isolated to its homogeneity has 100% affinity for DOPA. DOPA oxidative deaminase is functional at low concentrations of the substrate (< 100 micromol.L(-1)) and is independent of NADH. The molecular mass of the purified enzyme is approximately 190 kDa and the enzyme could be a pentamer of 54, 42, 34, 25, and 23 kDa subunits as determined by SDS-PAGE.
Subject(s)
Ammonia-Lyases/chemistry , Ammonia-Lyases/isolation & purification , Bacterial Proteins/chemistry , Bacterial Proteins/isolation & purification , Dihydroxyphenylalanine/metabolism , Rhodobacter sphaeroides/enzymology , Ammonia-Lyases/genetics , Ammonia-Lyases/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Kinetics , Molecular Weight , Oxidation-Reduction , Rhodobacter sphaeroides/chemistry , Rhodobacter sphaeroides/genetics , Substrate SpecificityABSTRACT
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 microM). The molecular mass of the purified enzyme was approximately 274kD and the enzyme could be a heterotetramer of 110, 82, 43 and 39kD subunits as determined by SDS-PAGE.