The role of the conserved residues His-246, His-199, and Tyr-255 in the catalysis of catechol 2,3-dioxygenase from Pseudomonas stutzeri OX1.
Catechol 2,3-dioxygenase (C2,3O) from Pseudomonas stutzeri OX1, which is able to grow on various aromatic substrates as the sole source of carbon and energy, has been expressed in Escherichia coli, purified, characterized, and found to be very similar to other dioxygenases from Pseudomonas species. Interestingly, the activity of the protein shows a rather unusual pH dependence when assayed on catechol. A model of the catalytic mechanism was developed that is able to reproduce the catalytic behavior of the protein as a function of the pH. The model includes multiple equilibria and four productive intermediates with different ionization states of the enzyme-substrate complex. The fitting of the theoretical curve to the experimental data suggests that a tyrosine and two histidine residues are involved in catalysis. Mutants (H246N)-, (H246A)-, (H199N)- and (Y255F)-C2,3O were produced to investigate the role of highly conserved His-199, His-246, and Tyr-255. The strongly reduced activity of the mutants suggests a primary catalytic role for each of these residues. Moreover, mutants at positions 199 and 246 display pH profiles different from that of the wild-type protein, thus indicating that residues His-246 and His-199 play a role in determining the unusual pH dependence of the enzyme. In addition, electron-withdrawing groups on catechol, which increase the acidity of the phenolic hydroxyl group, are able to counterbalance the effect of the mutation H246N in reducing catalytic activity but cause a further reduction of the activity of (H199N)-C2,3O. This finding suggests that His-246 is involved in the initial catechol deprotonation, whereas His-199 promotes the reaction between oxygen and the aromatic ring.
Viggiani
Ambra
A
Dipartimento di Chimica Biologica, Università di Napoli Federico II, Via Mezzocannone 16, 80134 Napoli, Italy.
Siani
Loredana
L
Notomista
Eugenio
E
Birolo
Leila
L
Pucci
Piero
P
Di Donato
Alberto
A
D001665:Binding Sites; D002384:Catalysis; D050557:Catechol 2,3-Dioxygenase; D002396:Catechols / Q000737:chemistry; D017124:Conserved Sequence; D049308:Dioxygenases / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D006639:Histidine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009154:Mutation; D010100:Oxygen / Q000737:chemistry; D044223:Pseudomonas stutzeri / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014443:Tyrosine / Q000737:chemistry*
D002396:Catechols; D007477:Ions; D014443:Tyrosine; D006639:Histidine; D049308:Dioxygenases; D050557:Catechol 2,3-Dioxygenase; C034221:catechol; D010100:Oxygen