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Titolo:
Functional analyses of Bph-Tod hybrid dioxygenase, which exhibits high degradation activity toward trichloroethylene
Autore:
Maeda, T; Takahashi, Y; Suenaga, H; Suyama, A; Goto, M; Furukawa, K;
Indirizzi:
Kyushu Univ, Fac Agr, Dept Biosci & Biotechnol, Appl Microbiol Lab, Fukuoka 8128581, Japan Kyushu Univ Fukuoka Japan 8128581 Microbiol Lab, Fukuoka 8128581, Japan
Titolo Testata:
JOURNAL OF BIOLOGICAL CHEMISTRY
fascicolo: 32, volume: 276, anno: 2001,
pagine: 29833 - 29838
SICI:
0021-9258(20010810)276:32<29833:FAOBHD>2.0.ZU;2-N
Fonte:
ISI
Lingua:
ENG
Soggetto:
PSEUDOMONAS-PSEUDOALCALIGENES KF707; B-356 BIPHENYL DIOXYGENASE; SITE-DIRECTED MUTAGENESIS; TOLUENE DIOXYGENASE; POLYCHLORINATED-BIPHENYLS; EFFICIENT DEGRADATION; SUBSTRATE-SPECIFICITY; OXIDIZING BACTERIUM; OXYGENASE COMPONENT; ESCHERICHIA-COLI;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
35
Recensione:
Indirizzi per estratti:
Indirizzo: Furukawa, K Kyushu Univ, Fac Agr, Dept Biosci & Biotechnol, Appl MicrobiolLab, Fukuoka 8128581, Japan Kyushu Univ Fukuoka Japan 8128581 ab, Fukuoka 8128581, Japan
Citazione:
T. Maeda et al., "Functional analyses of Bph-Tod hybrid dioxygenase, which exhibits high degradation activity toward trichloroethylene", J BIOL CHEM, 276(32), 2001, pp. 29833-29838

Abstract

Biphenyl dioxygenase (BphDox) in Pseudomonas pseudoalcaligenes KF707 is a multicomponent enzyme consisting of an iron-sulfur protein (ISP) that is composed of alpha (BphA1) and beta (BphA2) subunits, a ferredoxin (FDBphA3), and a ferredoxin reductase (FDRBphA4). A recombinant Escherichia coli strain expressing hybrid Dox that had replaced BphA1 with TodC1 (alpha subunit of toluene dioxygenase (TolDox) of Pseudomonas putida) exhibited high activity toward trichloroethylene (TCE) (Furukawa, K., Hirose, J., Hayashida, S.,and Nakamura, K. (1994) J. Bacteriol. 176, 2121-2123). In this study, ISP,FD, and FDR were purified and characterized. Reconstitution of the dioxygenase components consisting of purified ISPTodC1BphA2 FDBphA3 and FDRBphA4 exhibited oxygenation activities toward biphenyl, toluene, and TCE. Native polyacrylamide gel electrophoresis followed by the Ferguson plot analyses demonstrated that ISPTodC1BphA2 and ISPBphA1A2 were present as heterohexamers, whereas ISPTodC1C2 was present as a heterotetramer. The molecular activity (k(0)) of the hybrid Dox for TCE was 4.1 min(-1), which is comparable to that of TolDox. The K-m value of the hybrid Dox for TCE was 130 muM, which was lower than 250 muM for TolDox. These results suggest that the alpha subunit of ISP is crucial for the determination of substrate specificity and that the change in the alpha subunit conformation of ISP from alpha (2)beta (2) to alpha (3)beta (3), results in the acquisition of higher affinity to TCE, which may lead to high TCE degradation activity.

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Documento generato il 20/01/21 alle ore 03:05:45