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

Abstract

The tphA1 II and tphA2 II A3 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 II A3 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 Km values for TPA and the Vmax were determined to be 72+/-6 microM and 9.87+/-0.06 U/mg respectively.

Microbial degradation of phthalates: biochemistry and environmental implications

“…TPA is initially attacked by a 1,2‐dioxygenase yielding 2‐hydro‐ cis ‐1,2‐dihydroxy‐TPA (Schläfli et al ., ; Choi et al ., ; Fukuhara et al ., ), which can be directly decarboxylated by a cis ‐1,2‐dihydroxy dehydrogenase. Less is known about the ring‐hydroxylating oxygenase involved in IPA degradation.…”

Draft Genome Sequence of Comamonas sp. Strain E6 (NBRC 107749), a Degrader of Phthalate Isomers through the Protocatechuate 4,5-Cleavage Pathway

“…strain E6, isolated in Niigata, Japan, is a betaproteobacterial strain capable of degrading phthalate isomers, o -phthalate, terephthalate, and isophthalate ( 1 ), through the protocatechuate 4,5-cleavage (PCA45) pathway ( 2 ). In this strain, the functions and transcriptional regulations of tph , iph , and pmd genes responsible for the catabolism of terephthalate, isophthalate, and protocatechuate (PCA), respectively, have been characterized ( 1 , 3 – 7 ). In addition, a novel tripartite aromatic acid transporter (TAAT) involved in the uptake of terephthalate and isophthalate was discovered ( 8 ).…”

Biochemical Characterization of Inducible ‘Reductase’ Component of Benzoate Dioxygenase and Phthalate Isomer Dioxygenases from Pseudomonas aeruginosa strain PP4

“…The reductase component of phthalate dioxygenase from Pseudomonas cepacia DBO1 has been purified and structurally characterized [12][13][14]. The recombinant reductase component of terephthalate dioxygenase of Comamonas E6 has been purified and kinetically characterized [15]. To date, very few strains have been shown to degrade all three phthalate isomers as the sole source of carbon and energy.…”