title: Enzymatic properties of terephthalate 1,2-dioxygenase of Comamonas sp. 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.
authors.affiliation: Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Ngata, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Fukuhara
authors.forename: Daisuke
authors.initials: D
authors.lastname: Kasai
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Katayama
authors.forename: Masao
authors.initials: M
authors.lastname: Fukuda
authors.forename: Eiji
authors.initials: E
authors.lastname: Masai
mesh_terms: D020585:Comamonas / Q000235:genetics* / Q000378:metabolism; D049308:Dioxygenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004579:Electron Transport / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005288:Ferredoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D009243:NAD / Q000235:genetics / Q000378:metabolism; D009249:NADP / Q000235:genetics / Q000378:metabolism; D010795:Phthalic Acids; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D005288:Ferredoxins; D010795:Phthalic Acids; D011994:Recombinant Proteins; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; C011363:terephthalic acid; D049308:Dioxygenases; D005287:Ferredoxin-NADP Reductase