Evaluation of aromatic hydrocarbon decomposition catalyzed by the dioxygenase system and substitution of ferredoxin and ferredoxin reductase

• Published in: Environmental science and pollution research international Vol. 26; no. 33; pp. 34047 - 34057
• Main Authors: Yang, Jun Won, Cho, Wooyoun, Lim, Yejee, Park, Sungyoon, Lee, Dayoung, Jang, Hyun-A, Kim, Han S.
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01-11-2019; Springer Nature B.V
• Subjects: Alkanes; Appropriate Technologies to Combat Water Pollution; Aquatic Pollution; Aromatic hydrocarbons; Atmospheric Protection/Air Quality Control/Air Pollution; Binding sites; Biodegradation, Environmental; Camphor; Camphor 5-monooxygenase; Catalysis; Catalytic activity; Contaminants; Dehydrogenase; Dehydrogenases; Dehydrogenation; Dioxygenases - chemistry; Dioxygenases - metabolism; Earth and Environmental Science; Ecotoxicology; Environment; Environmental Chemistry; Environmental Health; Environmental Monitoring; Environmental science; Enzymes; Ferredoxin; Ferredoxin reductase; Ferredoxins - chemistry; Hydrocarbons; Hydroxylation; Kinetics; Monooxygenase; Oxidation-Reduction; Oxidoreductases - metabolism; Oxygenases - genetics; Oxygenases - metabolism; Proteins; Pseudomonas putida; Pseudomonas putida - metabolism; Reductases; Substrates; Sulfur; Toluene; Toluene - metabolism; Toluene dioxygenase; Waste Water Technology; Water Management; Water Pollution Control; Dioxygenase system; Catalytic activity; F1; Hill equation; Aromatic hydrocarbon contaminants; Substitute enzyme components; Pseudomonas putida F1
• ISSN: 0944-1344; 1614-7499
• DOI: 10.1007/s11356-018-3200-y

In this study, the catalytic activity and kinetic characteristics of the aromatic hydrocarbon dioxygenase system and the possibility of substituting its ferredoxin and ferredoxin reductase components were evaluated. The genes encoding toluene dioxygenase and toluene dihydrodiol dehydrogenase were cloned from
Pseudomonas putida
F1, and the corresponding enzymes were overexpressed and purified to homogeneity. Oxidative hydroxylation of toluene to
cis
-toluene dihydrodiol was catalyzed by toluene dioxygenase, and its subsequent dehydrogenation to 3-methylcatechol was catalyzed by toluene dihydrodiol dehydrogenase. The specific activity of the dioxygenase was 2.82 U/mg-protein, which is highly remarkable compared with the values obtained in previous researches conducted with crude extracts or insoluble forms of enzymes. Kinetic parameters, as characterized by the Hill equation, were
v
max
 = 497.2 μM/min,
K
M
 = 542.4 μM, and
n
H
 = 2.2, suggesting that toluene dioxygenase has at least three cooperative binding sites for toluene. In addition, the use of alternative ferredoxins and reductases was examined. Ferredoxin cloned from CYP153 could transfer electrons to the iron sulfur protein component of toluene dioxygenase. The ferredoxin could be reduced by ferredoxin, rubredoxin, and putidaredoxin reductases of CYP153, alkane-1 monooxygenase, and camphor 5-monooxygenase, respectively. The results provide useful information regarding the effective enzymatic biotreatment of hazardous aromatic hydrocarbon contaminants.