Enzymatic Properties of Terephthalate 1,2-Dioxygenase of Comamonas sp. Strain E6

Abstract

The tphA1_II and tphA2_IIA3_II genes of Comamonas sp. E6 perhaps code for the terephthalate (TPA) 1,2-dioxygenase (TPADO). To characterize E6 TPADO, these genes were expressed in a His-tagged form in Escherichia coli, and the recombinant proteins were purified. TPADO activity was reconstituted from TphA1_II and TphA2_IIA3_II, indicating that TPADO consists of a reductase (TphA1_II) and a terminal oxygenase component (TphA2_II and TphA3_II). TphA1_II contains FAD, and the presence of a plant-type [2Fe-2S] cluster was suggested. These results indicate that TPADO is a class IB aromatic ring-hydroxylating dioxygenase. NADH and NADPH were effective as electron donors for TphA1_II, but NADPH appeared to be the physiological electron donor, based on the kinetic parameters. TPADO showed activity only toward TPA, and Fe²⁺ was required for it. The K_m values for TPA and the V_max were determined to be 72±6 μM and 9.87±0.06 U/mg respectively.