Enzyme specificity of 2-nitrotoluene 2,3-dioxygenase from Pseudomonas sp. strain JS42 is determined by the C-terminal region of the alpha subunit of the oxygenase component.
Biotransformations with recombinant Escherichia coli expressing the genes encoding 2-nitrotoluene 2,3-dioxygenase (2NTDO) from Pseudomonas sp. strain JS42 demonstrated that 2NTDO catalyzes the dihydroxylation and/or monohydroxylation of a wide range of aromatic compounds. Extremely high nucleotide and deduced amino acid sequence identity exists between the components from 2NTDO and the corresponding components from 2,4-dinitrotoluene dioxygenase (2,4-DNTDO) from Burkholderia sp. strain DNT (formerly Pseudomonas sp. strain DNT). However, comparisons of the substrates oxidized by these dioxygenases show that they differ in substrate specificity, regiospecificity, and the enantiomeric composition of their oxidation products. Hybrid dioxygenases were constructed with the genes encoding 2NTDO and 2,4-DNTDO. Biotransformation experiments with these hybrid dioxygenases showed that the C-terminal region of the large subunit of the oxygenase component (ISP alpha) was responsible for the enzyme specificity differences observed between 2NTDO and 2,4-DNTDO. The small subunit of the terminal oxygenase component (ISP beta) was shown to play no role in determining the specificities of these dioxygenases.
Parales
J V
JV
Department of Microbiology and the Center for Biocatalysis and Bioprocessing, The University of Iowa, Iowa City 52242, USA.
Parales
R E
RE
Resnick
S M
SM
Gibson
D T
DT
D000595:Amino Acid Sequence; D001483:Base Sequence; D001673:Biodegradation, Environmental; D001711:Biotransformation; D049308:Dioxygenases; D004926:Escherichia coli / Q000235:genetics; D006900:Hydroxylation; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism; D009281:Naphthalenes / Q000378:metabolism; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D013237:Stereoisomerism; D013379:Substrate Specificity
D009097:Multienzyme Complexes; D009281:Naphthalenes; D011994:Recombinant Proteins; C031721:naphthalene; D010105:Oxygenases; D049308:Dioxygenases; C100896:2,4-dinitrotoluene dioxygenase; C102756:2-nitrotoluene dioxygenase; C038703:naphthalene dioxygenase