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

被引:68
作者
CERDAN, P [1 ]
WASSERFALLEN, A [1 ]
REKIK, M [1 ]
TIMMIS, KN [1 ]
HARAYAMA, S [1 ]
机构
[1] UNIV GENEVA, CTR MED, DEPT BIOCHEM MED, CH-1211 GENEVA 4, SWITZERLAND
关键词
D O I
10.1128/jb.176.19.6074-6081.1994
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
Catechol 2,3-dioxygenase encoded by TOL plasmid pWWO 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 K-m 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.
引用
收藏
页码:6074 / 6081
页数:8
相关论文
共 27 条