Construction of hybrid xylE genes between the two duplicate homologous genes from TOL plasmid pWW53: comparison of the kinetic properties of the gene products.
The two xylE genes for catechol 2,3-oxygenase, encoded by TOL plasmid pWW53, carry a common SalI restriction site within the reading frame. Each gene was cut at the SalI site and the 5' end of each gene spliced to the 3' end of the other to form hybrid genes, from both of which catalytically active catechol 2,3-oxygenase activities were expressed. The kinetic parameters were determined for the gene products of both the hybrid and the wild-type xylE genes with catechol, 3-methylcatechol and 4-methylcatechol as substrates. Comparison of the results suggested firstly, that the C-terminal regions of the enzymes determined both the binding and the catalytic specificity, and, secondly, that the N-terminal region of one of the enzymic gene products contained a secondary binding site which caused inhibition by excess substrate for methylcatechol substrates but not for catechol. One of the wild-type enzymes appeared to have an intrinsically higher activity for all three substrates than the other. This higher activity depended on the presence of both its C- and N-terminal regions, and in both hybrid enzymes, which contained only one of these regions, activity was significantly reduced.
Williams
P A
PA
School of Biological Sciences, University of Wales, Bangor, Gwynedd, UK.
Assinder
S J
SJ
Shaw
L E
LE
D001665:Binding Sites; D050557:Catechol 2,3-Dioxygenase; D002396:Catechols / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific; D049308:Dioxygenases; D005798:Genes, Bacterial; D007700:Kinetics; D010105:Oxygenases / Q000096:biosynthesis / Q000235:genetics*; D010957:Plasmids; D011549:Pseudomonas / Q000235:genetics*; D011995:Recombination, Genetic; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity
D002396:Catechols; D010105:Oxygenases; D049308:Dioxygenases; D050557:Catechol 2,3-Dioxygenase; D015252:Deoxyribonucleases, Type II Site-Specific; C029792:GTCGAC-specific type II deoxyribonucleases; C034221:catechol