Purification, characterization, and crystallization of the components of the nitrobenzene and 2-nitrotoluene dioxygenase enzyme systems.
The protein components of the 2-nitrotoluene (2NT) and nitrobenzene dioxygenase enzyme systems from Acidovorax sp. strain JS42 and Comamonas sp. strain JS765, respectively, were purified and characterized. These enzymes catalyze the initial step in the degradation of 2-nitrotoluene and nitrobenzene. The identical shared reductase and ferredoxin components were monomers of 35 and 11.5 kDa, respectively. The reductase component contained 1.86 g-atoms iron, 2.01 g-atoms sulfur, and one molecule of flavin adenine dinucleotide per monomer. Spectral properties of the reductase indicated the presence of a plant-type [2Fe-2S] center and a flavin. The reductase catalyzed the reduction of cytochrome c, ferricyanide, and 2,6-dichlorophenol indophenol. The ferredoxin contained 2.20 g-atoms iron and 1.99 g-atoms sulfur per monomer and had spectral properties indicative of a Rieske [2Fe-2S] center. The ferredoxin component could be effectively replaced by the ferredoxin from the Pseudomonas sp. strain NCIB 9816-4 naphthalene dioxygenase system but not by that from the Burkholderia sp. strain LB400 biphenyl or Pseudomonas putida F1 toluene dioxygenase system. The oxygenases from the 2-nitrotoluene and nitrobenzene dioxygenase systems each had spectral properties indicating the presence of a Rieske [2Fe-2S] center, and the subunit composition of each oxygenase was an alpha(3)beta(3) hexamer. The apparent K(m) of 2-nitrotoluene dioxygenase for 2NT was 20 muM, and that for naphthalene was 121 muM. The specificity constants were 7.0 muM(-1) min(-1) for 2NT and 1.2 muM(-1) min(-1) for naphthalene, indicating that the enzyme is more efficient with 2NT as a substrate. Diffraction-quality crystals of the two oxygenases were obtained.
Section of Microbiology, University of California, Davis, California 95616, USA. reparales@ucdavis.edu
R E
RE
Parales
R
R
Huang
C-L
CL
Yu
J V
JV
Parales
F K N
FK
Lee
D J
DJ
Lessner
M M
MM
Ivkovic-Jensen
W
W
Liu
R
R
Friemann
S
S
Ramaswamy
D T
DT
Gibson
D042621:Comamonadaceae / Q000201:enzymology* / Q000254:growth & development; D020585:Comamonas / Q000201:enzymology* / Q000254:growth & development; D003460:Crystallization; D049308:Dioxygenases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D009578:Nitrobenzenes / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014050:Toluene / Q000031:analogs & derivatives* / Q000378:metabolism*
D009578:Nitrobenzenes; D014050:Toluene; C036077:nitrobenzene; D049308:Dioxygenases