Ranjith N K, Sasikala Ch, Ramana Ch V
Department of Plant Sciences, School of Life Sciences, University of Hyderabad, P.O. Central University, Hyderabad 500 046, India.
Res Microbiol. 2007 Jul-Aug;158(6):506-11. doi: 10.1016/j.resmic.2007.04.008. Epub 2007 May 29.
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.
球形红杆菌OU5利用L-苯丙氨酸作为唯一的氮源进行生长。利用液相色谱-质谱联用仪（LC-MS）鉴定了L-苯丙氨酸分解代谢的代谢产物，即4-羟基苯丙氨酸（L-酪氨酸）、3,4-二羟基苯丙氨酸（多巴）、3,4-二羟基苯丙酮酸（DOPP）、3,4-二羟基苯乳酸（DOPLA）、3,4-二羟基苯乙酸（DOPAc）和3,4-二羟基苯甲酸（PC）。以2-氧代戊二酸作为氨基受体，在无细胞提取物中观察到多巴转氨酶活性，并通过质谱分析确认了产物DOPP。在没有2-氧代戊二酸的情况下也会发生多巴的还原性脱氨反应，其产物是3,4-二羟基苯丙酸（DPPA）和氨。将多巴还原性脱氨酶（DOPARDA）纯化至同质并对其进行了表征。DOPARDA对NADH有绝对需求，并且在低浓度底物（<150 microM）下具有活性。经SDS-PAGE测定，纯化酶的分子量约为274kD，该酶可能是由110、82、43和39kD亚基组成的异源四聚体。
