Expression, purification and substrate specificities of 3-nitrotoluene dioxygenase from Diaphorobacter sp. strain DS2.
3-Nitotoluene dioxygenase (3-NTDO) is the first enzyme in the degradation pathway of 3-nitrotoluene (3-NT) by Diaphorobacter sp. strain DS2. The complete gene sequences of 3-NTDO were PCR amplified from genomic DNA of Diaphorobacter sp., cloned, sequenced and expressed. The 3-NTDO gene revealed a multi component structure having a reductase, a ferredoxin and two oxygenase subunits. Clones expressing the different subunits were constructed in pET21a expression vector system and overexpressed in E. coli BL21(DE3) host. Each subunit was individually purified separately to homogeneity. The active recombinant enzyme was reconstituted in vitro by mixing all three purified subunits. The reconstituted recombinant enzyme could catalyse biotransformations on a variety of organic aromatics.
Department of Chemistry, Indian Institute of Technology, Kanpur 208016, India.
Deepak
D
Singh
Department of Chemistry, Indian Institute of Technology, Kanpur 208016, India.
Archana
A
Kumari
Institute of Stem Cell Biology and Regenerative Medicine, Bangalore, India.
S
S
Ramaswamy
Department of Chemistry, Indian Institute of Technology, Kanpur 208016, India. Electronic address: gurunath@iitk.ac.in.
Gurunath
G
Ramanathan
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D042621:Comamonadaceae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D004136:Dinitrobenzenes / Q000737:chemistry / Q000378:metabolism; D049308:Dioxygenases / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005288:Ferredoxins / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006841:Hydrocarbons, Aromatic / Q000737:chemistry / Q000378:metabolism; D015394:Molecular Structure; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014050:Toluene / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D004136:Dinitrobenzenes; D005288:Ferredoxins; D006841:Hydrocarbons, Aromatic; D021122:Protein Subunits; D011994:Recombinant Proteins; C029956:3-nitrotoluene; D014050:Toluene; C016403:2,4-dinitrotoluene; D010088:Oxidoreductases; D010105:Oxygenases; D049308:Dioxygenases
3-Nitotoluene dioxygenase (3-NTDO); Biodegradation; Diaphorobacter; Nitrotoluene (NT); Rieske dioxygenase