Substrate specificity differences between two catechol 2,3-dioxygenases encoded by the TOL and NAH plasmids from Pseudomonas putida.
The substrate specificities of two catechol 2,3-dioxygenases, one encoded by xylE on the TOL plasmid pWW0 and the other encoded by nahH on the NAH7 plasmid, were investigated. The XylE catechol 2,3-dioxygenase catalyzes the ring-cleavage of catechol, 3-methylcatechol and 4-methylcatechol. The NahH catechol 2,3-dioxygenase was partially deficient in oxidizing 3-methylcatechol due to defects in two catalytic properties. First, NahH has a lower kcat value for 3-methylcatechol compared to XylE, and secondly, NahH is more susceptible than XylE to suicide inhibition by 3-methylcatechol. To identify the amino acid residues of XylE and NahH responsible for the differences in the efficacy of the 3-methylcatechol oxidation, kcat and kinact (the rate constant for suicide inhibition) for 3-methylcatechol were determined for several NahH-XylE hybrid proteins, each of which consisted of the NahH sequence in the N-terminal region and the XylE sequence in the C-terminal region. It is shown that a single amino acid substitution present in the NahH sequence, His250-->Gln, was responsible for the reduced kcat and increased kinact values for 3-methylcatechol. In addition to the substitution at residue 250, some substitution(s) at residues 77-102 were responsible for the twofold difference in the kinact values for NahH and XylE with 3-methylcatechol. We also show that the binding site of 3-methylcatechol for suicide inhibition is different from the catalytic site.
Cerdan
P
P
Department of Medical Biochemistry, University Medical Center, Geneva, Switzerland.
Rekik
M
M
Harayama
S
S
D000595:Amino Acid Sequence; D001665:Binding Sites; D050557:Catechol 2,3-Dioxygenase; D049308:Dioxygenases; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D008969:Molecular Sequence Data; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D016958:Pseudomonas putida / Q000235:genetics*; D013379:Substrate Specificity
D010105:Oxygenases; D049308:Dioxygenases; D050557:Catechol 2,3-Dioxygenase