Substrate specificity of catechol 2,3-dioxygenase encoded by TOL plasmid pWW0 of Pseudomonas putida and its relationship to cell growth.
Catechol 2,3-dioxygenase encoded by TOL plasmid pWW0 of Pseudomonas putida 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-ethylcatechol. Two mutants of catechol 2,3-dioxygenases (4ECR1 and 4ECR6) able to oxidize 4-ethylcatechol, one mutant (3MCS) which exhibits only weak activity toward 3-methylcatechol but retained the ability to cleave catechol and 4-methylcatechol, and one phenotypic revertant of 3MCS (3MCR) which had regained the ability to oxidize 3-methylcatechol were characterized by determining their Km and partition ratio (the ratio of productive catalysis to suicide catalysis). The amino acid substitutions 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 ratio for this substrate, whereas the two mutant enzymes, 4ECR1 and 4ECR6, had higher partition ratios for it. Similarly, mutant enzyme 3MCS had a lower partition ratio for 3-methylcatechol than that of 3MCR. Molecular oxygen was required for the inactivation of the wild-type enzyme by 4-ethylcatechol and of 3MCS by 3-methylcatechol, and the inactivated enzymes could be reactivated by incubation with FeSO4 plus ascorbic acid. The enzyme inactivation is thus most likely mechanism based and occurred principally by oxidation and/or removal of the ferrous ion in the catalytic center. In general, partition ratios for catechols lower than 18,000 did not support bacterial growth. A possible meaning of the critical value of the partition ratio is discussed.
Cerdan
P
P
Department of Medical Biochemistry, University Medical Center, Geneva, Switzerland.
Wasserfallen
A
A
Rekik
M
M
Timmis
K N
KN
Harayama
S
S
D001205:Ascorbic Acid / Q000494:pharmacology; D050557:Catechol 2,3-Dioxygenase; D002396:Catechols / Q000378:metabolism* / Q000494:pharmacology; D002455:Cell Division; D049308:Dioxygenases; D004793:Enzyme Reactivators; D005296:Ferrous Compounds / Q000494:pharmacology; D007501:Iron / Q000032:analysis; D007700:Kinetics; D009154:Mutation; D010100:Oxygen / Q000494:pharmacology; D010105:Oxygenases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics*; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D013379:Substrate Specificity
D002396:Catechols; D004793:Enzyme Reactivators; D005296:Ferrous Compounds; C031136:3-methylcatechol; C018599:4-methylcatechol; C020748:ferrous sulfate; D007501:Iron; D010105:Oxygenases; D049308:Dioxygenases; D050557:Catechol 2,3-Dioxygenase; C034221:catechol; D001205:Ascorbic Acid; D010100:Oxygen