SUBSTRATE-SPECIFICITY DIFFERENCES BETWEEN 2 CATECHOL 2,3-DIOXYGENASESENCODED BY THE TOL AND NAH PLASMIDS FROM PSEUDOMONAS-PUTIDA

The substrate specificities of two catechol 2,3-dioxygenases, one enco ded by xylE on the TOL plasmid pWWO and the other encoded by nahH on t he NAH7 plasmid, were investigated. The XylE catechol 2,3-dioxygenase catalyzes the ring-cleavage of catechol, 3-methylcatechol and 4-methyl catechol. 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 k(cat) value for 3-methylcatechol compared t o XylE, and secondly, NahH is more susceptible than XylE to suicide in hibition by 3-methylcatechol. To identify the amino acid residues of X ylE and NahH responsible for the differences in the efficacy of the 3- methylcatechol oxidation, k(cat) and k(inact) (the rate constant for s uicide inhibition) for 3-methylcatechol were determined for several Na hH-XylE hybrid proteins, each of which consisted of the NahH sequence in the N-terminal region and the XylE sequence in the C-terminal regio n. It is shown that a single amino acid substitution present in the Na hH sequence, His250-->Gln, was responsible for the reduced k(cat) and increased kintact values for 3-methylcatechol. In addition to the subs titution at residue 250, some substitution(s) at residues 77-102 were responsible for the twofold difference in the k(inact) 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 catalyt ic site.