SUBSTRATE-SPECIFICITY OF CATECHOL 2,3-DIOXYGENASE ENCODED BY TOL PLASMID PWWO OF PSEUDOMONAS-PUTIDA AND ITS RELATIONSHIP TO CELL-GROWTH

Catechol 2,3-dioxygenase encoded by TOL plasmid pWWO of Pseudomonas pu tida consists of four identical subunits, each containing one ferrous ion. The enzyme catalyzes ring cleavage of catechol, 3-methylcatechol, and 4-methylcatechol but shows only weak activity toward 4-ethylcatec hol. Two mutants of catechol 2,3-dioxygenases (4ECR1 and 4ECR6) able t o oxidize 4-ethylcatechol, one mutant (3MCS) which exhibits only weak activity toward 3-methylcatechol but retained the ability to cleave ca techol and 4-methylcatechol, and one phenotypic revertant of 3MCS (3MC R) which had regained the ability to oxidize 3-methylcatechol were cha racterized by determining their K-m and partition ratio (the ratio of productive catalysis to suicide catalysis). The amino acid substitutio ns in the four mutant enzymes were also identified by sequencing their structural genes. Wild-type catechol 2,3-dioxygenase was inactivated during the catalysis of 4-ethylcatechol and thus had a low partition r atio for this substrate, whereas the two mutant enzymes, 4ECR1 and 4EC R6, had higher partition ratios for it. Similarly, mutant enzyme 3MCS had a lower partition ratio for 3-methylcatechol than that of 3MCR. Mo lecular oxygen was required for the inactivation of the wild-type enzy me by 4-ethylcatechol and of 3MCS by 3-methylcatechol, and the inactiv ated enzymes could be reactivated by incubation with FeSO4 plus ascorb ic acid. The enzyme inactivation is thus most likely mechanism based a nd occurred principally by oxidation and/or removal of the ferrous ion in the catalytic center. In general, partition ratios for catechols l ower than 18,000 did not support bacterial growth. A possible meaning of the critical value of the partition ratio is discussed.