ENZYME SPECIFICITY OF 2-NITROTOLUENE 2,3-DIOXYGENASE FROM PSEUDOMONASSP. STRAIN JS42 IS DETERMINED BY THE C-TERMINAL REGION OF THE ALPHA-SUBUNIT OF THE OXYGENASE COMPONENT

Biotransformations with recombinant Escherichia coli expressing the ge nes encoding 2-nitrotoluene 2,3-dioxygenase (2NTDO) from Pseudomonas s p. strain JS42 demonstrated that 2NTDO catalyzes the dihydroxylation a nd/or monohydroxylation of a nide range of aromatic compounds, Extreme ly high nucleotide and deduced amino acid sequence identity exists bet ween 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 th e substrates oxidized by these dioxygenases show that they differ in s ubstrate specificity, regiospecificity, and the enantiomeric compositi ons of their oxidation products, Hybrid dioxygenases were constructed with the genes encoding 2NTDO and 2,4-DNTDO, Biotransformation experim ents with these hybrid dioxygenases showed that the C-terminal region of the large subunit of the oxygenase component (ISP alpha) was respon sible for the enzyme specificity differences observed between 2NTDO an d 2,4-DNTDO, The small subunit of the terminal oxygenase component (IS P beta) was shown to play no role in determining the specificities of these dioxygenases.